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Antidepressant effects of nicotine and fluoxetine in an animal model of depression induced by neonatal treatment with clomipramine
Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 39 – 46 www.elsevier.com/locate/pnpbp
Antidepressant effects of nicotine and fluoxetine in an animal model of depression induced by neonatal treatment with clomipramine Gonzalo Va´zquez-Palacios*, Herlinda Bonilla-Jaime, Javier Vela´zquez-Moctezuma Neuroscience Laboratory, Department of Reproductive Biology, Universidad Auto´noma Metropolitana-Iztapalapa, Me´xico City C.P. 09340. Me´xico Accepted 27 August 2004 Available online 8 October 2004
Abstract The association between smoking and depression has been widely investigated. Most of these reports suggest that nicotine (NIC) may act as an antidepressant. To examine the suggested antidepressant effect of nicotine and its possible interaction with the serotonergic system, we assessed the effect of nicotine and fluoxetine (FLX) in an animal model of depression induced by neonatal treatment with clomipramine (CLI) and submitted to the forced swim test (FST). Results corroborated that CLI-treated rats displayed higher levels of immobility. After the administration of nicotine (0.4 mg/kg sc) acutely (1 day), subchonically (7 days), and chronically (14 days), CLI-treated rats significantly reduced the immobility and increased swimming without affecting climbing. These effects were similar to the effects induced for subchronic and chronic administration of the antidepressant fluoxetine (5 mg/kg sc), a selective serotonin re-uptake inhibitor. However, fluoxetine failed to affect immobility when it was administered acutely. No synergism was observed when both drugs were administered simultaneously. The present results further corroborate the antidepressant action of nicotine and fluoxetine. The increase of swimming during the FST has been linked to an increase of serotonergic activity. Thus, it could be possible that the antidepressant action of nicotine is mediated by the serotonergic system. D 2004 Elsevier Inc. All rights reserved. Keywords: Animal models of depression; Antidepressants; Clomipramine; Fluoxetine; Forced swimming test; Nicotine
1. Introduction Epidemiological studies of smokers suggested a link between nicotine (NIC) and depression. Thus, recent reports document that (1) smokers are more likely to exhibit depressive symptoms; (2) people who have at one time reported being depressed are more likely to be smokers (3) individuals with a history of depression have a much harder time giving up smoking; (4) cessation of smokers is often followed by depression (Quattrocki et al., 2000; Brown et al., 2000). Furthermore, bupropion, which has been well established as an antidepressant, has also been shown to act as an aid to smoking cessation (Jorenby Abbreviations: CLI, Clomipramine; FST, Forced swim Test; mg, Milligrams; Sc, subcutaneous; NIC, Nicotine; FLX, Fluoxetine; Ss, Subjects. * Corresponding author. Tel./fax: +525 8044704. E-mail address: [email protected] (G. Va´zquez-Palacios). 0278-5846/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2004.08.008
et al., 1999). In addition, transdermal nicotine patches can improve mood, as determined by the Hamilton Rating Scale for Depression, in nonsmoking patients with major depression (Salin-Pascual et al., 1995). These observations are consistent with the hypothesis that nicotine has antidepressant properties. In addition, Semba et al. (1998) reported that animals treated with chronic nicotine show a significant reduction in the number of escape failures in a learned helplessness paradigm in rats. Indirect evidence for the involvement of nicotinic acetylcholine receptors in depression has been reported (Tizabi et al., 1999). They used a genetic animal model of depression known as the Flinders Sensitive Line rats. This study found that acute or chronic administration of nicotine significantly improved the performance of these rats in the forced swimming test (FST). Most of the treatments currently employed as antidepressants improve serotonergic transmission (Blier et al., 1987; Blier and Montigny, 1998). In addition, there is
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growing evidence for a bidirectional relationship between the nicotine and the serotonergic system (Seth et al., 2002). For example, nicotine induced a concentration-dependent increase in serotonin release from rat midbrain slices which was accompanied by both increases and decreases of firing rates of dorsal raphe nucleus neurons firing rate (Mihailescu et al., 1998). Serotonin release was much higher during the decrease in firing rates, which suggests an action of nicotine on presynaptic receptors of serotonin neurons (Mihailescu et al., 1998). Therefore, it could be possible that antidepressant effect of nicotine involves serotonergic transmission. The rat FST is widely used to determine if pharmacological compounds exhibit antidepressant activity (Porsolt et al., 1977; Borsini and Meli, 1988; Detke et al., 1995). It has been hypothesized that immobility observed in the FST is a reliable marker of a state of anergia, lowered mood, or hopelessness (Porsolt et al., 1977, 1978; Velazquez-Moctezuma et al., 1993). More recently, it has been suggested that the immobility observed during the FST is reflecting depressive characteristics such as those observed during arrested flight or entrapment (Dixon, 1998; Dixon and Fisch, 1998; Gilbert and Allan, 1998; Fullilove, 2002). The FST in rats is sensitive to a wide range of antidepressants (Borsini and Meli, 1988). It has been recently reported that a modification of the scoring procedure allows swimming and climbing to be measured; these behaviors are claimed to be selectively associated with serotonergic and noradrenergic activity (Detke et al., 1995; Lucki, 1997). One commonly used method to produce rats with behavioral changes consistent with human depression is the neonatal administration of clomipramine (CLI), a monoamine re-uptake inhibitor (Vogel et al., 1990a). These rats exhibit behavioral abnormalities resembling endogenous depression when they reach adulthood (Vogel et al., 1990a). These abnormalities include reduced aggressiveness (Vijayakumar and Meti, 1999; Martinez-Gonzalez et al., 2002), decreased pleasure seeking behaviors (Vogel et al., 1990b), diminished sexual activity (Bonilla-Jaime et al., 1998; Vogel et al., 1996), shorter REM sleep onset, and more REM sleep periods (Frank and Heller, 1997; Vogel et al., 1990c). In addition, it has been reported also that CLItreated rats show neurochemical alterations in the serotonergic system (Yavari et al., 1993; Hansen and Mikkelsen, 1998; Feenstra et al., 1996; Andersen et al., 2002). Some behavioral abnormalities in these rats (sexual, aggressive, and motor) begin to normalize after antidepressant treatments such as administration of imipramine or after REM sleep deprivation (Vogel et al., 1990a). It has been reported that neonatal CLI treatment results in an exaggerated immobility in the FST compared to their control with saline neonatal treatment (Bonilla-Jaime et al., 1998; VelazquezMoctezuma et al., 1993). In order to assess the suggested antidepressant effect of nicotine and its possible interaction with the serotonergic system, both nicotine and fluoxetine were administered in
an animal model of depression induced by neonatal treatment with CLI and submitted to the FST.
2. Materials and methods 2.1. Animals Pregnant Wistar rats from our own vivarium were used in this study. Three days after delivery, female pups were eliminated from this study and male pups were crossfostered, keeping the same size (N=5) in all the litters. All animals were kept on a reversed 12-h lighting cycle with lights on at 2100 h and controlled temperature. Food and water were available ad libitum. Male pups were injected subcutaneously twice a day (0900 and 1800) from 8 to 21 days of age. Each litter received the same treatment. One group (CLI, N=170) received clomipramine (15 mg/kg 0.1 ml) in each injection. A control group (CON, N=170) received saline in the same volume and the same number of injections. There was also an additional control group of intact rats (N=10). At 23 days of age, the pups were weaned and housed in groups (five per cage with the same treatment) until 4 months of age. All housing and behavioral procedures conform to the Principles of Laboratory Animal Care issued by the National Institutes of Health. 2.2. Forced swimming test (FST) The procedure for the FST used has previously been described (Detke et al., 1995) and was very similar to that described by Porsolt (1977, 1978), except that the water was deeper (Detke and Lucki, 1996). Rats were placed in individual glass cylinders (40 cm tall20 cm in diameter) containing 30 cm of water, maintained at 23–25 8C, so the rats could not support themselves by touching the bottom with their feet. Two swimming sessions were conducted between 1100 and 1600 h: an initial 15-min pretest followed 24 h later by a 5-min test. Following both swimming sessions, the rats were removed from cylinders, dried with towels, and returned to their home cages. Test sessions were videotaped for later scoring. The water in the cylinders was changed after every other trial to avoid confounding results because of possible alarm substances from urine or feces. A time-sampling technique was employed to score several behaviors during a single viewing. This method has previously been described and shown to be reliable and valid for detecting the effects of different antidepressant drugs (Detke et al., 1995). At the end of each 5-s period during the 5-min test, the scorer rated the rat’s behavior as one of the following behavioral categories: (1) the animal was judged to be immobile whenever it remained floating passively in the water and only making those movements necessary to keep its head above the water; (2) swimming was defined as the movements of the four extremities that
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allow the rat to move around or cross the cylinder; and (3) climbing was defined as active movements with forepaws in and out of the water, usually directed against the walls. An experienced evaluator who was blind to the treatment conditions did all of the behavioral scoring. Scores for each behavior were expressed as the total number of counts per 5min session. The numbers of counts represent the number of times that a specified behavior of immobility, swimming, or climbing was observed during the 5-min test.
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2.4. Data analysis Statistical analysis for immobility, swimming, and climbing data was performed using a factorial ANOVA (224) where neonatal treatment (saline vs. CLI), treatment duration (1, 7, 14, and 21 days), and treatments (saline, NIC, FLX, and NIC+FLX) were the factors. When significant, ANOVA was followed by the Dunn post hoc test to determine the source of significance.
2.3. Pharmacological treatments 3. Results CLI-treated rats and saline-treated control rats were randomly assigned to the following groups and treatments (N=10 in each group): Acute treatment: Group A1: One subcutaneous (sc) injection of nicotine bitartrate (0.4 mg/kg bw) in a volume of 0.2 ml of saline solution. The NIC injection was given 10 min before the start of the 5-min test of the FST. Group A2: One sc injection of FLX HCl (5 mg/kg bw) in a volume of 0.2 ml of distilled water. FLX injection was given 40 min before the start of the 5-min FST. Group A3: Acute administration of combined FLX and NIC. Drugs were administered in combination in the same dose and at the times mentioned above. Group A4: Control. Subjects (Ss) received one sc injection of 0.2 ml of saline 20 min before the 5-min FST. Drug doses were selected on the basis of previous reports where the effectiveness in the FST swim tests was demonstrated. In addition, no increase of general locomotor activity has been reported for these doses (Tizabi et al., 1999; Detke et al., 1995). Subchronic administration: Group B1: Ss received sc NIC administration for 7 days. Group B2: Ss received sc administration of FLX for 7 days. Group B3: Ss received sc administration of both drugs for 7 days. Group B4: Control. Ss received sc injection of saline for 7 days. The 5-min FST test was done on the last day of treatment for all of the subchronic groups. Chronic treatment: Group C1: Ss received sc NIC injections for 14 days. Group C2: Ss received sc FLX injections for 14 days. Group C3: Ss received sc administration of both drugs for 14 days. Group B4: Control. Ss received sc injection of saline for 7 days. The 5-min FST test was done on the last day of treatment for all of the chronic groups. Postchronic withdrawal: Group D1: Ss received sc administration of NIC for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D2: Ss received sc administration of FLX for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D3: Ss received sc administration of FLX and nicotine for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D4: Ss received sc injections of saline for 14 days and were submitted to the FST 7 days after termination of the injections.
Concerning immobility significant differences were detected in neonatal treatments [ F(1,312)=43.93; pb0.0001], days of treatment [ F(3,272)=17.41; pb0.0001], and drugs [ F(3,288)=35.81; pb0.0001]. Regarding swimming, there were also differences in neonatal treatments [ F(1,312)= 30.97; pb0.0001], days of treatment [ F(3,272)=14.96; pb0.0001], and drugs [ F(3,288)=42.5; pb0.0001]. No significant differences were detected concerning climbing in neonatal treatment [ F(1,312)=3.81; pb0.519], days of treatment [ F (3,272)=2.97; p b0.321], and drugs [ F(3,288)=2.95; pb0.329]. The baseline behavior of CLI-treated, saline, and intact groups in the FST is shown in Fig. 1. At adulthood, the CLItreated group exhibited significantly more immobility [ F(2,27)=23.17, pb0.0001; Dunn pb0.01) accompanied by a significant decrease in swimming behavior [ F(2,27)= 19.36, pb0.0001; Dunn pb0.01) when compared to both intact rats and neonatal rats treated with saline solution. The climbing behavior in CLI-treated rats was not affected [ F(2,27)=4.7, pb0.095]. Fig. 2 shows the effects of acute treatments on behaviors in the FST. In rats neonatally treated with clomipramine,
Fig. 1. Amount of immobility, swimming, and climbing behaviors in rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Neonatal administration of clomipramine (CLI), neonatal administration of saline solution (SALINE) and intact rats (INTACT). N in each group=10. ANOVA followed by the Dunn test *=pb0.01 compared to SALINE control; o=pb0.01 compared to INTACT.
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11.21; pb0.0001; Dunn pb0.01]. This was accompanied by a significant increase in swimming behavior [ F(3,288)=3.4; pb0.018; Dunn pb0.01] without modifying the frequency of climbing behavior [ F(3,288)=1.16; pb0.32]. FLX treatment for 7 days also decreased the frequency of immobility counts [ F(3,315)=4.55; pb0.0001; Dunn pb0.01] and increased the swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.01], but there was no significant difference in climbing counts [ F(3,315)=1.84; pb0.61]. Combined FLX+ NIC administration also reduced immobility [ F(3,315)= 4.55; pb0.0001; Dunn pb0.01] and increased swimming [ F(3,315)=4.55; pb0.0001; Dunn pb0.01]. This was similar when compared to the effects obtained after the individual administration of NIC or FLX. Rats neonatally treated with saline showed a similar response to the different treatments [ F(3,288)=1.35; pb0.2568]. Fig. 4 shows the effects of 14 days of treatment on behaviors in the FST. The effect of NIC was similar to that
Fig. 2. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Acute (1 day) administration of saline solution (CON), nicotine (NIC); 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=pb0.05; **=pb0.01, compared to control.
NIC significantly decreased the frequency of immobility counts during the 5-min test session [ F(3,288)=21.35; pb0.0001; Dunn pb0.01]. This was accompanied by a significant increase in swimming behavior [ F(3,288)=3.4; pb0.018; Dunn pb0.01], but there was no significant difference in climbing behavior after nicotine [ F(3,288)=1.16; pb0.32]. Note that FLX acute administration did not induced significant changes when compared to the control group. The combined FLX+NIC induced a significant reduction of immobility behavior [ F(3,288)=21.35; pb0.0001; Dunn pb0.01] with an increase in the frequency of swimming counts [ F(3,288)=3.4; pb0.018; Dunn pb0.05]. These effects were similar to those induced after the acute administration of NIC. In the rats neonatally treated with saline, the drugs induced the same response observed in the CLI-treated rats [ F(3,288)=1.35; pb2568]. The effects of subchronic treatments are shown in Fig. 3. NIC administration significantly decreased the frequency of imobility counts during the 5-min test session [ F(3,288)=
Fig. 3. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Subchronic (7 days) administration of saline solution (CON), nicotine (NIC); 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=0.05; **=pb0.01 compared to control.
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When Ss were submitted to the FST 7 days after the termination of all treatments, no significant differences were observed when compared to their respective controls, neither in the group of rats neonatally treated with saline nor in the rats neonatally treated with clomipramine [ F(3,288)=1.35; pb0.2568].
4. Discussion
Fig. 4. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Chronic (14 days) administration of saline solution (CON), nicotine (NIC), 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=pb0.05; **=pb0.01 compared to control.
obtained after acute and subchronic administration. The frequency of immobility counts decreased [ F(3,315)=4.55; pb0.0001; Dunn pb0.05] and was accompanied by a significant increase in swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.05], while climbing was not affected [ F(3,315)=1.84; pb0.61]. FLX treatment induced a reduction in the frequency of immobility counts during the 5-min test session [ F(3,315)=4.55; pb0.0001; Dunn pb0.05]. This was accompanied by a significant increase in swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.01), but there was no significant difference in climbing behavior [ F(3,315)=4.55; pb1.84; Dunn pb0.61]. Combined administration of FLX+NIC also showed a similar effect to that observed after 7 days of treatment, with a reduction of immobility [ F(3,315)=4.55; pb0.0001; Dunn pb0.01) and an increase of swimming [ F(3,315)=4.55; pb0.0001; Dunn pb0.01]. Rats neonatally treated with saline showed a similar response to the different treatments.
The present results corroborate that neonatal treatment with clomipramine induces an exaggerated immobility in the FST swimming test when animals reach adult age (+25%; Bonilla-Jaime et al., 1998; Velazquez-Moctezuma et al., 1993). This was accompanied by a significant decrease in swimming behavior ( 52.63%) without modifying climbing behavior counts. These data suggest that CLItreated rats have altered serotonergic system activity. The alteration of serotonergic system after neonatal treatment with serotonin re-uptake blockers has been repeatedly reported. Neonatally CLI-treated rats exhibit a decrease in the firing rate of the serotonergic units located in the dorsal raphe nucleus (Yavari et al., 1993). Rats neonatally treated with CLI or with the serotonin selective re-uptake inhibitor LU 10-134-C show a decrease of the 5-HT transporter mRNA expression, also in the raphe nucleus (Hansen and Mikkelsen, 1998). CLI neonatal treatment also induces a decrease of the hypothalamic levels of 5-HT (Feenstra et al., 1996), while other authors reported a significant decrease of both 5-HT and noradrenaline levels in the brain stem, septum, hippocampus, hypothalamus, and frontal cortex (Vijayakumar and Meti, 1999). Neonatal CLItreated rats also show lower levels of serotonin and dopamine in the limbic regions of the left side, while control animals have lower levels in the right side (Andersen et al., 2002). Furthermore, the neonatal administration of the selective serotonin re-uptake inhibitor Lu 10-134-C induces an increase of immobility in the FST similar to that observed after CLI neonatal treatment (Hansen et al., 1997). The present study also provides new evidence for the antidepressant-like effects of nicotine. Assuming that the FST is a reliable tool to detect antidepressant activity, the present results show that NIC has antidepressant effects that can be observed following its acute and chronic administration. Inasmuch as NIC only modified swimming levels in the FST, it could be assumed that nicotinic effects have a serotonergic component that is also present from the first administration because the serotonergic system has been implicated in swimming (Detke et al., 1995, 1997; Page et al., 1999). NIC maximum effect was reached after 7 days of treatment, and there was a return to control levels 7 days after the treatment. Although the effects of NIC withdrawal after chronic treatment have been reported (Watkins et al., 2000), the assessment of these effects has been done in the short-term. Our results suggest that 7 days after the ending of NIC treatment, the behavioral effects are not longer observed.
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FLX also showed an antidepressant effect in CLItreated rats. However, FLX seemed to require repeated administration to initiate its effects on immobility in the FST. Its maximum effect was reached after 7 days of treatment and remained the same after 14 days. The effect disappeared when the chronic treatment was suspended. Combined administration of FLX/NIC did not seem to have synergistic actions. Both acute NIC and FLX/NIC treatments induced a decrease of immobility that reached basal frequencies observed for both intact rats and neonatal rats treated with saline. The results obtained in the present study are quite similar to those previously reported by our group in intact rats (Vazquez-Palacios et al., 2004). NIC treatment showed an immediate antidepressant-like effect on the FST, while the effect of FLX was observed only after its repeated administration. It is possible that NIC administration induces an immediate release of 5-HT in an amount capable of modifying the depressant-like behavior, while FLX administration takes a while to induce levels of 5-HT in an amount needed to induce behavioral changes. The present results are consistent with preliminary clinical reports showing antidepressant effects of NIC when it is applied transdermally (Salin-Pascual et al., 1995). Furthermore, NIC also induced antidepressant-like activity in the FST when it was administered to Flinders Sensitive Line rats after acute and chronic treatment (Tizabi et al., 1999). Flinders rats have been selectively bred for cholinergic supersensitivity, and they have been proposed as an animal model of depression (Overstreet, 2002). Chronic NIC exposure induced a significant reduction in the number of escape failures in the learned helplessness paradigm (Semba et al., 1998). These effects also have been reversed with SIB-1508 Y treatment, a selective agonist of nicotinic receptors (Ferguson et al., 2000). The rapid antidepressants effects of NIC showed in the FST are in contrast to what is reported classically as to the therapeutic effects in depressed humans. Most of the antidepressants used in humans seem to require repetitive administration for at least 10 days in order to be effective (Thompson, 2002). The rapid antidepressant action showed by NIC opens the possibility that it can be used during the first days of treatment before the onset of the therapeutic effects of a classical antidepressant. Nicotine/serotonin interactions have been extensively demonstrated (Seth et al., 2002). NIC induced a dosedependent increase in the release of serotonin from rat midbrain slices, which was accompanied by both increases and decreases of firing rates of neurons located in the dorsal raphe nucleus (Mihailescu et al., 1998). Serotonin release was much higher during the decrease in firing rates, which suggest an action of nicotine on presynaptic receptors of serotonin neurons (Mihailescu et al., 1998). In anaesthetized rats, systemic administration of NIC induced a transient inhibition of most neurons recorded in the DRN. This inhibition was blocked by a selective 5-HT1A receptor
antagonist WAY-1000635, indicating that NIC might influence 5-HT1A receptors (Engberg et al., 2000). It has been reported that chronic, but not acute, administration of selective serotonin inhibitors, such as FLX, induces antidepressant effects in the FST (Kelly and Leonard, 1994; Detke et al., 1995; Re´ne´ric and Lucki, 1998). It has also been indicated that a single injection of FLX induced only a transient increase of extracellular serotonin while repeated administration significantly increased base line levels of serotonin (Kreiss and Lucki, 1995; Rutter et al., 1994). In addition, the chronic administration of FLX also seems to induce a desensitization of the 5-HT1A receptors (Li et al., 1996; Raap et al., 1999). This receptor is particularly important in mediating nicotine-induced behaviors (File et al., 2000). Because NIC alters serotonergic transmission, it has been suggested that NIC modulates the expression of 5HT1A receptors located in certain cortical and limbic regions involved in the etiology of depression (Kenny et al., 2001; Kennedy et al., 2001; Mayberg et al., 2000; Stockmeier, 1997). These data support the notion that NIC administration can have a positive influence on the antidepressant treatment with serotonergic agents.
5. Conclusion This study further supports the notion that nicotine is able to exert an antidepressant action not only in animal models of depression but also in normal rats. The results obtained in the FST swim test suggest a serotonergic mediation of the antidepressant action of nicotine. However, the lack of synergy observed when fluoxetine was coadministered does not support this notion. Thus, the mediation of the antidepressant effect of nicotine remains to be elucidated.
Acknowledgments The authors wish to express their gratitude to Dr. David W. Krogmann and Edith Monroy for their helpful comments on the manuscript. GVP is recipient of a scholarship from the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT), and the present study is part of his Doctoral Degree in Experimental Biology. The study was partly supported by CONACyT 2002-C01-42307 to JVM.
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Antidepressant effects of nicotine and fluoxetine in an animal model of depression induced by neonatal treatment with clomipramine Gonzalo Va´zquez-Palacios*, Herlinda Bonilla-Jaime, Javier Vela´zquez-Moctezuma Neuroscience Laboratory, Department of Reproductive Biology, Universidad Auto´noma Metropolitana-Iztapalapa, Me´xico City C.P. 09340. Me´xico Accepted 27 August 2004 Available online 8 October 2004
Abstract The association between smoking and depression has been widely investigated. Most of these reports suggest that nicotine (NIC) may act as an antidepressant. To examine the suggested antidepressant effect of nicotine and its possible interaction with the serotonergic system, we assessed the effect of nicotine and fluoxetine (FLX) in an animal model of depression induced by neonatal treatment with clomipramine (CLI) and submitted to the forced swim test (FST). Results corroborated that CLI-treated rats displayed higher levels of immobility. After the administration of nicotine (0.4 mg/kg sc) acutely (1 day), subchonically (7 days), and chronically (14 days), CLI-treated rats significantly reduced the immobility and increased swimming without affecting climbing. These effects were similar to the effects induced for subchronic and chronic administration of the antidepressant fluoxetine (5 mg/kg sc), a selective serotonin re-uptake inhibitor. However, fluoxetine failed to affect immobility when it was administered acutely. No synergism was observed when both drugs were administered simultaneously. The present results further corroborate the antidepressant action of nicotine and fluoxetine. The increase of swimming during the FST has been linked to an increase of serotonergic activity. Thus, it could be possible that the antidepressant action of nicotine is mediated by the serotonergic system. D 2004 Elsevier Inc. All rights reserved. Keywords: Animal models of depression; Antidepressants; Clomipramine; Fluoxetine; Forced swimming test; Nicotine
1. Introduction Epidemiological studies of smokers suggested a link between nicotine (NIC) and depression. Thus, recent reports document that (1) smokers are more likely to exhibit depressive symptoms; (2) people who have at one time reported being depressed are more likely to be smokers (3) individuals with a history of depression have a much harder time giving up smoking; (4) cessation of smokers is often followed by depression (Quattrocki et al., 2000; Brown et al., 2000). Furthermore, bupropion, which has been well established as an antidepressant, has also been shown to act as an aid to smoking cessation (Jorenby Abbreviations: CLI, Clomipramine; FST, Forced swim Test; mg, Milligrams; Sc, subcutaneous; NIC, Nicotine; FLX, Fluoxetine; Ss, Subjects. * Corresponding author. Tel./fax: +525 8044704. E-mail address: [email protected] (G. Va´zquez-Palacios). 0278-5846/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2004.08.008
et al., 1999). In addition, transdermal nicotine patches can improve mood, as determined by the Hamilton Rating Scale for Depression, in nonsmoking patients with major depression (Salin-Pascual et al., 1995). These observations are consistent with the hypothesis that nicotine has antidepressant properties. In addition, Semba et al. (1998) reported that animals treated with chronic nicotine show a significant reduction in the number of escape failures in a learned helplessness paradigm in rats. Indirect evidence for the involvement of nicotinic acetylcholine receptors in depression has been reported (Tizabi et al., 1999). They used a genetic animal model of depression known as the Flinders Sensitive Line rats. This study found that acute or chronic administration of nicotine significantly improved the performance of these rats in the forced swimming test (FST). Most of the treatments currently employed as antidepressants improve serotonergic transmission (Blier et al., 1987; Blier and Montigny, 1998). In addition, there is
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growing evidence for a bidirectional relationship between the nicotine and the serotonergic system (Seth et al., 2002). For example, nicotine induced a concentration-dependent increase in serotonin release from rat midbrain slices which was accompanied by both increases and decreases of firing rates of dorsal raphe nucleus neurons firing rate (Mihailescu et al., 1998). Serotonin release was much higher during the decrease in firing rates, which suggests an action of nicotine on presynaptic receptors of serotonin neurons (Mihailescu et al., 1998). Therefore, it could be possible that antidepressant effect of nicotine involves serotonergic transmission. The rat FST is widely used to determine if pharmacological compounds exhibit antidepressant activity (Porsolt et al., 1977; Borsini and Meli, 1988; Detke et al., 1995). It has been hypothesized that immobility observed in the FST is a reliable marker of a state of anergia, lowered mood, or hopelessness (Porsolt et al., 1977, 1978; Velazquez-Moctezuma et al., 1993). More recently, it has been suggested that the immobility observed during the FST is reflecting depressive characteristics such as those observed during arrested flight or entrapment (Dixon, 1998; Dixon and Fisch, 1998; Gilbert and Allan, 1998; Fullilove, 2002). The FST in rats is sensitive to a wide range of antidepressants (Borsini and Meli, 1988). It has been recently reported that a modification of the scoring procedure allows swimming and climbing to be measured; these behaviors are claimed to be selectively associated with serotonergic and noradrenergic activity (Detke et al., 1995; Lucki, 1997). One commonly used method to produce rats with behavioral changes consistent with human depression is the neonatal administration of clomipramine (CLI), a monoamine re-uptake inhibitor (Vogel et al., 1990a). These rats exhibit behavioral abnormalities resembling endogenous depression when they reach adulthood (Vogel et al., 1990a). These abnormalities include reduced aggressiveness (Vijayakumar and Meti, 1999; Martinez-Gonzalez et al., 2002), decreased pleasure seeking behaviors (Vogel et al., 1990b), diminished sexual activity (Bonilla-Jaime et al., 1998; Vogel et al., 1996), shorter REM sleep onset, and more REM sleep periods (Frank and Heller, 1997; Vogel et al., 1990c). In addition, it has been reported also that CLItreated rats show neurochemical alterations in the serotonergic system (Yavari et al., 1993; Hansen and Mikkelsen, 1998; Feenstra et al., 1996; Andersen et al., 2002). Some behavioral abnormalities in these rats (sexual, aggressive, and motor) begin to normalize after antidepressant treatments such as administration of imipramine or after REM sleep deprivation (Vogel et al., 1990a). It has been reported that neonatal CLI treatment results in an exaggerated immobility in the FST compared to their control with saline neonatal treatment (Bonilla-Jaime et al., 1998; VelazquezMoctezuma et al., 1993). In order to assess the suggested antidepressant effect of nicotine and its possible interaction with the serotonergic system, both nicotine and fluoxetine were administered in
an animal model of depression induced by neonatal treatment with CLI and submitted to the FST.
2. Materials and methods 2.1. Animals Pregnant Wistar rats from our own vivarium were used in this study. Three days after delivery, female pups were eliminated from this study and male pups were crossfostered, keeping the same size (N=5) in all the litters. All animals were kept on a reversed 12-h lighting cycle with lights on at 2100 h and controlled temperature. Food and water were available ad libitum. Male pups were injected subcutaneously twice a day (0900 and 1800) from 8 to 21 days of age. Each litter received the same treatment. One group (CLI, N=170) received clomipramine (15 mg/kg 0.1 ml) in each injection. A control group (CON, N=170) received saline in the same volume and the same number of injections. There was also an additional control group of intact rats (N=10). At 23 days of age, the pups were weaned and housed in groups (five per cage with the same treatment) until 4 months of age. All housing and behavioral procedures conform to the Principles of Laboratory Animal Care issued by the National Institutes of Health. 2.2. Forced swimming test (FST) The procedure for the FST used has previously been described (Detke et al., 1995) and was very similar to that described by Porsolt (1977, 1978), except that the water was deeper (Detke and Lucki, 1996). Rats were placed in individual glass cylinders (40 cm tall20 cm in diameter) containing 30 cm of water, maintained at 23–25 8C, so the rats could not support themselves by touching the bottom with their feet. Two swimming sessions were conducted between 1100 and 1600 h: an initial 15-min pretest followed 24 h later by a 5-min test. Following both swimming sessions, the rats were removed from cylinders, dried with towels, and returned to their home cages. Test sessions were videotaped for later scoring. The water in the cylinders was changed after every other trial to avoid confounding results because of possible alarm substances from urine or feces. A time-sampling technique was employed to score several behaviors during a single viewing. This method has previously been described and shown to be reliable and valid for detecting the effects of different antidepressant drugs (Detke et al., 1995). At the end of each 5-s period during the 5-min test, the scorer rated the rat’s behavior as one of the following behavioral categories: (1) the animal was judged to be immobile whenever it remained floating passively in the water and only making those movements necessary to keep its head above the water; (2) swimming was defined as the movements of the four extremities that
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allow the rat to move around or cross the cylinder; and (3) climbing was defined as active movements with forepaws in and out of the water, usually directed against the walls. An experienced evaluator who was blind to the treatment conditions did all of the behavioral scoring. Scores for each behavior were expressed as the total number of counts per 5min session. The numbers of counts represent the number of times that a specified behavior of immobility, swimming, or climbing was observed during the 5-min test.
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2.4. Data analysis Statistical analysis for immobility, swimming, and climbing data was performed using a factorial ANOVA (224) where neonatal treatment (saline vs. CLI), treatment duration (1, 7, 14, and 21 days), and treatments (saline, NIC, FLX, and NIC+FLX) were the factors. When significant, ANOVA was followed by the Dunn post hoc test to determine the source of significance.
2.3. Pharmacological treatments 3. Results CLI-treated rats and saline-treated control rats were randomly assigned to the following groups and treatments (N=10 in each group): Acute treatment: Group A1: One subcutaneous (sc) injection of nicotine bitartrate (0.4 mg/kg bw) in a volume of 0.2 ml of saline solution. The NIC injection was given 10 min before the start of the 5-min test of the FST. Group A2: One sc injection of FLX HCl (5 mg/kg bw) in a volume of 0.2 ml of distilled water. FLX injection was given 40 min before the start of the 5-min FST. Group A3: Acute administration of combined FLX and NIC. Drugs were administered in combination in the same dose and at the times mentioned above. Group A4: Control. Subjects (Ss) received one sc injection of 0.2 ml of saline 20 min before the 5-min FST. Drug doses were selected on the basis of previous reports where the effectiveness in the FST swim tests was demonstrated. In addition, no increase of general locomotor activity has been reported for these doses (Tizabi et al., 1999; Detke et al., 1995). Subchronic administration: Group B1: Ss received sc NIC administration for 7 days. Group B2: Ss received sc administration of FLX for 7 days. Group B3: Ss received sc administration of both drugs for 7 days. Group B4: Control. Ss received sc injection of saline for 7 days. The 5-min FST test was done on the last day of treatment for all of the subchronic groups. Chronic treatment: Group C1: Ss received sc NIC injections for 14 days. Group C2: Ss received sc FLX injections for 14 days. Group C3: Ss received sc administration of both drugs for 14 days. Group B4: Control. Ss received sc injection of saline for 7 days. The 5-min FST test was done on the last day of treatment for all of the chronic groups. Postchronic withdrawal: Group D1: Ss received sc administration of NIC for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D2: Ss received sc administration of FLX for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D3: Ss received sc administration of FLX and nicotine for 14 days and were submitted to the FST 7 days after termination of the treatment. Group D4: Ss received sc injections of saline for 14 days and were submitted to the FST 7 days after termination of the injections.
Concerning immobility significant differences were detected in neonatal treatments [ F(1,312)=43.93; pb0.0001], days of treatment [ F(3,272)=17.41; pb0.0001], and drugs [ F(3,288)=35.81; pb0.0001]. Regarding swimming, there were also differences in neonatal treatments [ F(1,312)= 30.97; pb0.0001], days of treatment [ F(3,272)=14.96; pb0.0001], and drugs [ F(3,288)=42.5; pb0.0001]. No significant differences were detected concerning climbing in neonatal treatment [ F(1,312)=3.81; pb0.519], days of treatment [ F (3,272)=2.97; p b0.321], and drugs [ F(3,288)=2.95; pb0.329]. The baseline behavior of CLI-treated, saline, and intact groups in the FST is shown in Fig. 1. At adulthood, the CLItreated group exhibited significantly more immobility [ F(2,27)=23.17, pb0.0001; Dunn pb0.01) accompanied by a significant decrease in swimming behavior [ F(2,27)= 19.36, pb0.0001; Dunn pb0.01) when compared to both intact rats and neonatal rats treated with saline solution. The climbing behavior in CLI-treated rats was not affected [ F(2,27)=4.7, pb0.095]. Fig. 2 shows the effects of acute treatments on behaviors in the FST. In rats neonatally treated with clomipramine,
Fig. 1. Amount of immobility, swimming, and climbing behaviors in rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Neonatal administration of clomipramine (CLI), neonatal administration of saline solution (SALINE) and intact rats (INTACT). N in each group=10. ANOVA followed by the Dunn test *=pb0.01 compared to SALINE control; o=pb0.01 compared to INTACT.
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11.21; pb0.0001; Dunn pb0.01]. This was accompanied by a significant increase in swimming behavior [ F(3,288)=3.4; pb0.018; Dunn pb0.01] without modifying the frequency of climbing behavior [ F(3,288)=1.16; pb0.32]. FLX treatment for 7 days also decreased the frequency of immobility counts [ F(3,315)=4.55; pb0.0001; Dunn pb0.01] and increased the swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.01], but there was no significant difference in climbing counts [ F(3,315)=1.84; pb0.61]. Combined FLX+ NIC administration also reduced immobility [ F(3,315)= 4.55; pb0.0001; Dunn pb0.01] and increased swimming [ F(3,315)=4.55; pb0.0001; Dunn pb0.01]. This was similar when compared to the effects obtained after the individual administration of NIC or FLX. Rats neonatally treated with saline showed a similar response to the different treatments [ F(3,288)=1.35; pb0.2568]. Fig. 4 shows the effects of 14 days of treatment on behaviors in the FST. The effect of NIC was similar to that
Fig. 2. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Acute (1 day) administration of saline solution (CON), nicotine (NIC); 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=pb0.05; **=pb0.01, compared to control.
NIC significantly decreased the frequency of immobility counts during the 5-min test session [ F(3,288)=21.35; pb0.0001; Dunn pb0.01]. This was accompanied by a significant increase in swimming behavior [ F(3,288)=3.4; pb0.018; Dunn pb0.01], but there was no significant difference in climbing behavior after nicotine [ F(3,288)=1.16; pb0.32]. Note that FLX acute administration did not induced significant changes when compared to the control group. The combined FLX+NIC induced a significant reduction of immobility behavior [ F(3,288)=21.35; pb0.0001; Dunn pb0.01] with an increase in the frequency of swimming counts [ F(3,288)=3.4; pb0.018; Dunn pb0.05]. These effects were similar to those induced after the acute administration of NIC. In the rats neonatally treated with saline, the drugs induced the same response observed in the CLI-treated rats [ F(3,288)=1.35; pb2568]. The effects of subchronic treatments are shown in Fig. 3. NIC administration significantly decreased the frequency of imobility counts during the 5-min test session [ F(3,288)=
Fig. 3. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Subchronic (7 days) administration of saline solution (CON), nicotine (NIC); 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=0.05; **=pb0.01 compared to control.
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When Ss were submitted to the FST 7 days after the termination of all treatments, no significant differences were observed when compared to their respective controls, neither in the group of rats neonatally treated with saline nor in the rats neonatally treated with clomipramine [ F(3,288)=1.35; pb0.2568].
4. Discussion
Fig. 4. Amount of immobility, swimming, and climbing behaviors in CLItreated rats sampled every 5 s during the 5-min period in the forced swimming test. Bars represent the mean number of counts over the 5 min of the test (+/ S.E.M.). Chronic (14 days) administration of saline solution (CON), nicotine (NIC), 0.4mg/kg/day), fluoxetine (FLX; 5 mg/kg/day), and the combination of FLX plus NIC (FLX+NIC). N in each group=10. Upper panel: rats neonatally treated with saline. Lower panel: rats neonatally treated with clomipramine. ANOVA followed by the Dunn test *=pb0.05; **=pb0.01 compared to control.
obtained after acute and subchronic administration. The frequency of immobility counts decreased [ F(3,315)=4.55; pb0.0001; Dunn pb0.05] and was accompanied by a significant increase in swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.05], while climbing was not affected [ F(3,315)=1.84; pb0.61]. FLX treatment induced a reduction in the frequency of immobility counts during the 5-min test session [ F(3,315)=4.55; pb0.0001; Dunn pb0.05]. This was accompanied by a significant increase in swimming behavior [ F(3,315)=4.4; pb0.0001; Dunn pb0.01), but there was no significant difference in climbing behavior [ F(3,315)=4.55; pb1.84; Dunn pb0.61]. Combined administration of FLX+NIC also showed a similar effect to that observed after 7 days of treatment, with a reduction of immobility [ F(3,315)=4.55; pb0.0001; Dunn pb0.01) and an increase of swimming [ F(3,315)=4.55; pb0.0001; Dunn pb0.01]. Rats neonatally treated with saline showed a similar response to the different treatments.
The present results corroborate that neonatal treatment with clomipramine induces an exaggerated immobility in the FST swimming test when animals reach adult age (+25%; Bonilla-Jaime et al., 1998; Velazquez-Moctezuma et al., 1993). This was accompanied by a significant decrease in swimming behavior ( 52.63%) without modifying climbing behavior counts. These data suggest that CLItreated rats have altered serotonergic system activity. The alteration of serotonergic system after neonatal treatment with serotonin re-uptake blockers has been repeatedly reported. Neonatally CLI-treated rats exhibit a decrease in the firing rate of the serotonergic units located in the dorsal raphe nucleus (Yavari et al., 1993). Rats neonatally treated with CLI or with the serotonin selective re-uptake inhibitor LU 10-134-C show a decrease of the 5-HT transporter mRNA expression, also in the raphe nucleus (Hansen and Mikkelsen, 1998). CLI neonatal treatment also induces a decrease of the hypothalamic levels of 5-HT (Feenstra et al., 1996), while other authors reported a significant decrease of both 5-HT and noradrenaline levels in the brain stem, septum, hippocampus, hypothalamus, and frontal cortex (Vijayakumar and Meti, 1999). Neonatal CLItreated rats also show lower levels of serotonin and dopamine in the limbic regions of the left side, while control animals have lower levels in the right side (Andersen et al., 2002). Furthermore, the neonatal administration of the selective serotonin re-uptake inhibitor Lu 10-134-C induces an increase of immobility in the FST similar to that observed after CLI neonatal treatment (Hansen et al., 1997). The present study also provides new evidence for the antidepressant-like effects of nicotine. Assuming that the FST is a reliable tool to detect antidepressant activity, the present results show that NIC has antidepressant effects that can be observed following its acute and chronic administration. Inasmuch as NIC only modified swimming levels in the FST, it could be assumed that nicotinic effects have a serotonergic component that is also present from the first administration because the serotonergic system has been implicated in swimming (Detke et al., 1995, 1997; Page et al., 1999). NIC maximum effect was reached after 7 days of treatment, and there was a return to control levels 7 days after the treatment. Although the effects of NIC withdrawal after chronic treatment have been reported (Watkins et al., 2000), the assessment of these effects has been done in the short-term. Our results suggest that 7 days after the ending of NIC treatment, the behavioral effects are not longer observed.
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FLX also showed an antidepressant effect in CLItreated rats. However, FLX seemed to require repeated administration to initiate its effects on immobility in the FST. Its maximum effect was reached after 7 days of treatment and remained the same after 14 days. The effect disappeared when the chronic treatment was suspended. Combined administration of FLX/NIC did not seem to have synergistic actions. Both acute NIC and FLX/NIC treatments induced a decrease of immobility that reached basal frequencies observed for both intact rats and neonatal rats treated with saline. The results obtained in the present study are quite similar to those previously reported by our group in intact rats (Vazquez-Palacios et al., 2004). NIC treatment showed an immediate antidepressant-like effect on the FST, while the effect of FLX was observed only after its repeated administration. It is possible that NIC administration induces an immediate release of 5-HT in an amount capable of modifying the depressant-like behavior, while FLX administration takes a while to induce levels of 5-HT in an amount needed to induce behavioral changes. The present results are consistent with preliminary clinical reports showing antidepressant effects of NIC when it is applied transdermally (Salin-Pascual et al., 1995). Furthermore, NIC also induced antidepressant-like activity in the FST when it was administered to Flinders Sensitive Line rats after acute and chronic treatment (Tizabi et al., 1999). Flinders rats have been selectively bred for cholinergic supersensitivity, and they have been proposed as an animal model of depression (Overstreet, 2002). Chronic NIC exposure induced a significant reduction in the number of escape failures in the learned helplessness paradigm (Semba et al., 1998). These effects also have been reversed with SIB-1508 Y treatment, a selective agonist of nicotinic receptors (Ferguson et al., 2000). The rapid antidepressants effects of NIC showed in the FST are in contrast to what is reported classically as to the therapeutic effects in depressed humans. Most of the antidepressants used in humans seem to require repetitive administration for at least 10 days in order to be effective (Thompson, 2002). The rapid antidepressant action showed by NIC opens the possibility that it can be used during the first days of treatment before the onset of the therapeutic effects of a classical antidepressant. Nicotine/serotonin interactions have been extensively demonstrated (Seth et al., 2002). NIC induced a dosedependent increase in the release of serotonin from rat midbrain slices, which was accompanied by both increases and decreases of firing rates of neurons located in the dorsal raphe nucleus (Mihailescu et al., 1998). Serotonin release was much higher during the decrease in firing rates, which suggest an action of nicotine on presynaptic receptors of serotonin neurons (Mihailescu et al., 1998). In anaesthetized rats, systemic administration of NIC induced a transient inhibition of most neurons recorded in the DRN. This inhibition was blocked by a selective 5-HT1A receptor
antagonist WAY-1000635, indicating that NIC might influence 5-HT1A receptors (Engberg et al., 2000). It has been reported that chronic, but not acute, administration of selective serotonin inhibitors, such as FLX, induces antidepressant effects in the FST (Kelly and Leonard, 1994; Detke et al., 1995; Re´ne´ric and Lucki, 1998). It has also been indicated that a single injection of FLX induced only a transient increase of extracellular serotonin while repeated administration significantly increased base line levels of serotonin (Kreiss and Lucki, 1995; Rutter et al., 1994). In addition, the chronic administration of FLX also seems to induce a desensitization of the 5-HT1A receptors (Li et al., 1996; Raap et al., 1999). This receptor is particularly important in mediating nicotine-induced behaviors (File et al., 2000). Because NIC alters serotonergic transmission, it has been suggested that NIC modulates the expression of 5HT1A receptors located in certain cortical and limbic regions involved in the etiology of depression (Kenny et al., 2001; Kennedy et al., 2001; Mayberg et al., 2000; Stockmeier, 1997). These data support the notion that NIC administration can have a positive influence on the antidepressant treatment with serotonergic agents.
5. Conclusion This study further supports the notion that nicotine is able to exert an antidepressant action not only in animal models of depression but also in normal rats. The results obtained in the FST swim test suggest a serotonergic mediation of the antidepressant action of nicotine. However, the lack of synergy observed when fluoxetine was coadministered does not support this notion. Thus, the mediation of the antidepressant effect of nicotine remains to be elucidated.
Acknowledgments The authors wish to express their gratitude to Dr. David W. Krogmann and Edith Monroy for their helpful comments on the manuscript. GVP is recipient of a scholarship from the Consejo Nacional de Ciencia y Tecnologı´a (CONACYT), and the present study is part of his Doctoral Degree in Experimental Biology. The study was partly supported by CONACyT 2002-C01-42307 to JVM.
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