Mirtazapine attenuates anxiety- and depression-like behaviors in rats during cocaine withdrawal

Abstract

Background:

Anxiety and depression, key symptoms of the cocaine withdrawal syndrome in human addicts, are considered the main factors that precipitate relapse in chronic cocaine addiction. Preclinical studies have found that rodents exposed to different withdrawal periods show an increase in anxiety and depressive-like behavior. Mirtazapine – a tetracyclic medication – is used primarily to treat depression and, sometimes, anxiety. It has also successfully improved withdrawal symptoms in drug-dependent patients.

Aim:

This study sought to determine whether chronic dosing of mirtazapine during cocaine withdrawal reduced depression- and anxiety-like behaviors that characterize cocaine withdrawal in animals.

Methods:

Cocaine pre-treated Wistar rats were subjected to a 60-day cocaine withdrawal period during which depression- and anxiety-like behaviors were evaluated in open field tests (OFT), the elevated plus-maze (EPM), the light–dark box test (LDT), the forced swimming test (FST) and spontaneous locomotor activity (SLA).

Results:

We found that chronic dosing with different doses of mirtazapine (30 and 60 mg/kg) decreased depression- and anxiety-like behaviors induced by different doses of cocaine (10, 20 and 40 mg/kg) during the 60-day cocaine withdrawal.

Interpretation:

Our results suggest that the pharmacological effect of mirtazapine on its target sites of action (α₂-adrenergic and 5-HT_2A and 5-HT₃ receptors) within the brain may improve depression- and anxiety-like behaviors for long periods.

Conclusion:

Therefore, the findings support the use of mirtazapine as a potentially effective therapy to reduce anxiety and depressive-like behavior during cocaine withdrawal.

Keywords

Cocaine withdrawal mirtazapine depression-like behavior anxiety-like behavior antidepressant

Introduction

Abuse of illegal drugs is one of the most serious health problems in the world, with cocaine among the most widely used psychostimulants (Grant et al., 2016). Cocaine abuse usually results in major physical and mental health problems. Like other drugs of abuse, cocaine induces both powerful positive reinforcing effects and negative affective states that appear when the drug is withdrawn (El-Hage et al., 2012).

Cocaine withdrawal causes changes in the brain at the molecular, cellular and morphological levels as well as in behavior (Yang et al., 2017). In addition, it produces a wide range of adverse psychological and physical symptoms, including sleep disturbances, dysphoria, depression, anxiety, insomnia, changes in appetite, psychomotor retardation, increased sensitivity to drug-associated environmental cues and cocaine-craving (Gawin, 1991). Several studies have suggested that persistent adverse emotional and cognitive symptoms contribute to the chronic and relapsing nature of cocaine addiction (Ladrón de Guevara-Miranda et al., 2017). Moreover, mood and anxiety disorders are highly prevalent among cocaine addicts (Araos et al., 2014, 2015; Conway et al., 2006; Grant et al., 2004; Pedraz et al., 2015a, 2015b).

Research has shown that at the neurochemical level cocaine withdrawal brings about a decrease in noradrenaline (NA), dopamine (DA) or serotonin (5-HT) release. This has been associated with severe anxiety and depression (Dworkin et al., 1995), which are the more characteristic symptoms of cocaine withdrawal and appear within its first days (Walsh et al., 2009).

Anxiety and depression associated with drug withdrawal have been effectively treated with anxiolytics and antidepressants (Kampman et al., 2006). Chronic use of some medications to treat depression such as fluoxetine, however, causes serious adverse effects (Hodes et al., 2010; Olivier et al., 2011).

Mirtazapine, a tetracyclic antidepressant, is used primarily to treat depression, though it is also sometimes used for anxiety or sleeplessness and other indications (de Boer, 1995, 1996; Hashimoto et al., 2016; Tsutsumi et al., 2016).

A recent meta-analysis (Cipriani et al., 2009) suggested that mirtazapine may demonstrate a stronger effect for decreasing depressive symptoms in addition to other medications including selective serotonin reuptake inhibitors (SSRIs) (Croom et al., 2009; Watanbe et al., 2008). In addition, clinical trials have demonstrated that mirtazapine effectively treats various types of anxiety disorders (Gambi et al., 2005; Goodnick et al., 1999; Schutters et al., 2010); significantly improves symptoms of depression, anxiety and insomnia; and minimizes physical and subjective discomfort and symptoms of dysphoria during benzodiazepine, alcohol and cocaine withdrawal (Afshar et al., 2012; Chandrasekaran, 2008; Liappas et al., 2003, 2004).

Some authors have suggested that the negative emotional states that characterize drug withdrawal are associated with an individual’s urgent need to resume drug use (Rudoy and Van-Bockstaele, 2007). They have also surmised that the decrease in adverse cocaine withdrawal symptoms may reduce relapses (de Oliveira Citó Mdo et al., 2012). Since preclinical studies and clinical trials have proven mirtazapine’s effectiveness in decreasing not only cocaine behavioral effects (Afshar et al., 2012; Barbosa-Méndez et al., 2017a; Salazar-Juárez et al., 2016) but also benzodiazepine and alcohol withdrawal symptoms (Chandrasekaran, 2008; Liappas et al., 2003, 2004), it would then be important to determine if chronic dosing of mirtazapine during cocaine withdrawal reduces the depression- and anxiety-like behaviors, which characterize cocaine withdrawal in animals.

To that end, our study used various tests. These included open field (OFT), the elevated plus-maze (EPM), the light–dark box test (LDT), and the forced swimming test (FST), which we used together with spontaneous locomotor activity (SLA) as behavioral tools to determine the effect of mirtazapine on anxiety- and depression-like behaviors (Bahi, 2016; Crawley and Goodwin, 1980; Pellow and File, 1986; Porsolt et al., 2001) at different points during withdrawal. We found that chronic dosing of mirtazapine (30 mg/kg) decreased depression- and anxiety-like behaviors during a 60-day period of cocaine withdrawal.

Experimental procedures

Animals

We used male Wistar rats that weighed 250–280 g at the beginning of the study. They were housed four per cage in standard plastic rodent cages (57 × 35 × 20 cm) in a colony room maintained at 21 ± 2°C and at 40–50% humidity under a 12-h light/dark cycle (lights on at 7:00 hours). The animals had free access to water and rodent chow pellets, except during experimental sessions.

Because the cocaine effects and the expression of several components of the dopaminergic system are more potent during the illumination phase (first hours of the day), all the experiments were conducted during the light phase, between 9:00 and 15:00 hours. The study procedures were approved by the Committees on Bioethics and Institutional Laboratory Animal Care and Use, in strict compliance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health.

Drugs

Cocaine hydrochloride was kindly donated by the Mexican government under strict regulatory controls. All the drugs used in experimental animals were kept under official surveillance (COFEPRIS-LC-0004-2003). Cocaine hydrochloride and mirtazapine (REMERON, Schering-Plough-Organon-SANFER) were dissolved in sterile saline solution (0.9% NaCl, Sigma Aldrich). Both solutions were freshly prepared before intraperitoneal (i.p.) administration. During the experiments, the solutions were maintained at −20°C. Saline (0.9% NaCl) was used as control in all experiments. The volume injected into each animal depended on its body weight (BW): BW (g)/100 mL. To avoid tissue damage to the injection area, new needles (25G) per animal/session were used. In addition, veterinarians monitored the injection area daily, to verify the presence of hardening, bulging, sinking, bleeding or redness of the skin.

Dose selection

For this study, the optimal mirtazapine dose (30 mg/kg) was determined in accordance with previous reports. They showed that ⩾30 mg/kg mirtazapine does not affect SLA (Salazar-Juárez et al., 2016) or produce sedation, nor does it induce weight gain (Bittolo et al., 2016; Salazar-Juárez et al., 2017), in rats.

Experimental procedures

The study used 800 male Wistar rats divided into three groups, and each group underwent a different experiment. For Experiment 1, we used 160 animals further divided into four experimental groups (n = 40; Figure 1(a)); for Experiments 2 and 3, we used 320 animals assigned to eight groups (n = 40; Figures 2(a) and 3(a)). For each experimental group, OFT (n = 8), EPM (n = 8), LDT (n = 8), FST (n = 8) and SLA (n = 8) were performed at days 1, 15, 30, and 60.

Experiment 1

To determine whether mirtazapine (30 mg/kg) reduced anxiety- and depression-like behaviors during cocaine withdrawal up to 60 days’ duration, Experiment 1 included two phases. These were cocaine induction (phase I), which lasted 10 days, and cocaine withdrawal (phase II), which lasted 30 days (Figure 1(a)).

The saline (SAL + SAL), the mirtazapine (MIR + SAL) and the cocaine (SAL + COC) groups received saline solution (9% NaCl, i.p.) or cocaine (10 mg/kg, i.p.) during the induction phase. During the cocaine withdrawal phase, the SAL + SAL and the MIR + SAL groups received saline solution and mirtazapine (30 mg/kg, i.p.), respectively, and the SAL + COC group received only saline daily.

The cocaine + mirtazapine groups (MIR + COC-_1d, MIR + COC-_15d, MIR + COC-_30d and MIR + COC-_60d) received cocaine daily during induction. In the cocaine withdrawal phase, the rats received a fixed dose of mirtazapine (30 g/kg, i.p; Figure 1(a)).

The animals of the groups SAL + COC, MIR + COC-_1d, MIR + COC-_15d, MIR + COC-_30d and MIR + COC-_60d received the administration of cocaine once a day for 10 days during the induction phase.

For each experimental group, OFT (n = 8), EPM (n = 8), LDT (n = 8), FST (n = 8) and SLA (n = 8) were performed at days 1, 15, 30 and 60 after cocaine withdrawal. Thirty minutes after mirtazapine administration, anxiety and depressive-like behavior were assessed and recorded for each animal.

Experiment 2

Experiment 2 evaluated the effect of different doses of mirtazapine on the anxiety- and depression-like behaviors during cocaine withdrawal, as per the protocol described in Experiment 1.

The SAL (SAL + SAL) and the COC (SAL + COC) groups were administered the treatments described in Experiment 1. The MIR groups (MIR-_15mg + SAL, MIR-_30mg + SAL and MIR-_60mg + SAL) received mirtazapine in different doses (15, 30 and 60 mg/kg, i.p., respectively).

The cocaine + mirtazapine groups (MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC) received cocaine (10 mg/kg, i.p.) daily during induction. In the cocaine withdrawal phase, the rats received mirtazapine (15, 30 and 60 mg/kg, i.p., respectively; Figure 2(a)).

For each experimental group, anxiety- and depressive-like behavior testing was performed at days 1, 15 and 30 after cocaine withdrawal. Thirty minutes after mirtazapine administration, anxiety- and depression-like behaviors were assessed and recorded for each animal.

Experiment 3

Experiment 3 characterized the effect of a fixed dose of mirtazapine (30 mg/kg) on the anxiety- and depression-like behaviors during cocaine withdrawal induced by different doses of cocaine as the protocol described in Experiment 1.

The SAL (SAL + SAL) and the MIR (MIR + SAL) groups were administered the same treatments used in Experiment 1. The cocaine groups (SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg) received cocaine in different doses (10, 20 and 40 mg/kg, i.p., respectively) in the induction phase. During cocaine withdrawal, the groups were administered saline only every day (Figure 3(a)).

The cocaine + mirtazapine groups (MIR +COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg) received cocaine daily in different doses (10, 20 and 40 mg/kg, i.p., respectively) during induction. In the cocaine withdrawal phase, the animals in these groups received mirtazapine (30 mg/kg, i.p.). Anxiety and depressive-like behavior tests were performed as described in Experiment 2. Thirty minutes after mirtazapine administration, anxiety- and depression-like behaviors were evaluated and recorded for each animal.

Apparatus

The FST has been validated and widely used to assess depression-like behavior in rodents (Cryan et al., 2005). Transparent Plexiglas (OMNIALVA, Instruments, Mexico) cylinders (25 cm in diameter, 63 cm high) filled with water (22–24 ± 0.5°C, 45 cm depth) were used during each session. A dark screen separated the cylinders. The water was replaced between subjects and the cylinders were cleaned after each testing session.

Procedure

The experiments were carried out according to the method of Porsolt et al. (1977a, 1977b, 1978). Each rat was gently placed in the transparent Plexiglas cylinder for 5 min. Immobility was recorded by a digital video camera.

Immobility (“depression-like behavior,” for the purposes of this study) was defined as floating passively and making only those movements necessary to keep its head and nose just above the water surface. Increased immobility in rodents subjected to the FST has been associated with depression. After each session, the rats were towel dried and returned to their home cages.

Statistical analysis

In Experiment 1, a three-way ANOVA was used, with groups (saline and mirtazapine), treatment (saline and cocaine) and cocaine withdrawal time (1, 15, 30 and 60 days) as the between subjects factor was performed to determine the differences in the anxiety- and depressive-like behavior. When a significant F value was found in the interaction, a post hoc analysis of differences in responses between groups was performed using an additional Tukey’s test. In Experiments 2 and 3, a four-way ANOVA was used with groups (saline and cocaine), treatment (saline and mirtazapine), treatment doses (cocaine or mirtazapine doses) and cocaine withdrawal time (1, 15, 30 and 60 days) as the between subjects factor, following a Tukey’s post hoc test.

The locomotor activity, during the induction phase, was analyzed by means of a two- or three-way ANOVA with groups (saline and cocaine), treatment (saline and mirtazapine) and treatment doses (cocaine or mirtazapine doses) as the between subjects factor, following a Tukey’s post hoc test.

All results were expressed as mean ± standard error (SEM). When a p-value was less than 0.05, we concluded that a statistically significant difference existed between experimental groups. Calculations were performed using STATISTICA software.

Results

Experiment 1: Mirtazapine reduced anxiety- and depression-like behaviors during cocaine withdrawal

Locomotor activity induction

As shown in Figure 1(a), dosing of cocaine (10 mg/kg) increased locomotor activity during induction (two-way ANOVA; in the groups by treatment interaction, F (1. 99) = 41.10, p < 0.0003) phase, compared with the SAL + SAL (p <0.001) and MIR + SAL (p <0.001) groups.

Anxiety-like behavior

Figure 2(b) to (f) and Table 1 shows a significant decrease in entries and time spent in the EPM open and close arms (Figure 2(b); Table 1), in time spent in the center, in number of entries into the center and periphery, and in rearing in the OFT (Figure 2(c); Table 1), as well as entries and time spent in the light and dark compartment in the LDT (Figure 2(d); Table 1) shown by the SAL + COC group compared with the SAL + SAL and MIR + SAL groups at 1 (p < 0.002), 15 (p < 0.001), 30 (p < 0.001) and 60 (p < 0.001) days of cocaine withdrawal (Table 2).

Table 1.

Comparisons between the groups, treatments and cocaine withdrawal time by means of a three-way ANOVA during cocaine withdrawal in the behavioral parameters evaluated with EPM, OFT, LDT, FST and LSA.

Experiment 1	EPM				OFT				LDT				FST	SLA
Three-way ANOVA	OAEN	OAPT	CAEN	CAPT	CEN	CPT	PPT	RRG	LEN	LPT	DEN	DPT	IT	LAC
(A) Groups F (1, 84) =	33.26, p < 0.003	27.68, p < 0.004	103.56, p < 0.001	103.56, p < 0.001	42.87, p < 0.002	94.36, p < 0.002	103.56, p < 0.001	103.56, p < 0.001	48.69, p < 0.003	64.18, p < 0.001	103.56, p < 0.001	103.56, p < 0.001	109.44, p < 0.001	61.78, p < 0.004
(B) Treatments F (1, 84) =	103.56, p < 0.001	215.78, p < 0.001	138.45, p < 0.003	197.87, p < 0.003	90.55, p < 0.002	165.14, p < 0.002	81.79, p < 0.001	106.13, p < 0.003	278.69, p < 0.003	405.36, p < 0.001	135.01, p < 0.03	580.68, p < 0.002	59.91, p < 0.001	188.71, p < 0.004
(C) Cocaine-withdrawal time F (3, 84) =	18.79, p < 0.003	56.41, p < 0.003	11.19, p < 0.002	14.50, p < 0.003	38.97, p < 0.004	44.68, p < 0.002	70.42, p < 0.02	41.89, p < 0.03	134.55, p < 0.002	77.59, p < 0.001	176.51, p < 0.001	93.34, p < 0.001	24.58, p < 0.004	106.42, p < 0.003
A × B × C interactions F (3, 84) =	20.52, p < 0.002	83.95, p < 0.001	94.34, p < 0.002	85.53, p < 0.001	80.09, p < 0.002	137.97, p < 0.001	93.34, p < 0.003	146.08, p < 0.002	223.11, p < 0.001	317.72, p < 0.001	255.97, p < 0.02	186.79, p < 0.03	16.92, p < 0.003	144.88, p < 0.001

Table 2.

Comparisons between the groups by means of a Tukey test during the 1, 15, 30 and 60 days of cocaine withdrawal.

Experiment 1	p <	EPM			OFT			LDT
TIME	Group	OAPT	CAEN	CAPT	CPT	PPT	RRG	LPT	DEN	DPT
1 day	SAL+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	SAL+COC	0.002*	NS	NS	0.002*	0.002*	NS	0.002*	0.03*	0.02*
	MIR+SAL	0.002 ^#	0.001 ^#	0.003 ^#	0.002 ^#	0.001 ^#	0.003 ^#	0.002 ^#	0.001 ^#	0.001 ^#
	MIR+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
15 days	SAL+SAL	0.002 ⁺	0.04 ⁺	0.03 ⁺	0.002 ⁺	0.002 ⁺	0.001 ⁺	0.002 ⁺	0.02 ⁺	0.02 ⁺
	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.002*	0.003*	0.001*	0.001*	0.002*
	MIR+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	MIR+COC	0.001**	0.002**	0.003**	0.001**	0.002**	0.004**	0.001**	0.001**	0.001**
30 days	SAL+SAL	0.002 ⁺	0.003 ⁺	0.002 ⁺	0.002 ⁺	0.001 ⁺	0.001 ⁺	0.002 ⁺	0.003 ⁺	0.003 ⁺
	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.002*	0.002*
	MIR+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	MIR+COC	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**
60 days	SAL+SAL	0.002 ⁺	0.001 ⁺	0.002 ⁺	0.002 ⁺	0.001 ⁺	0.001 ⁺	0.002 ⁺	0.001 ⁺	0.001 ⁺
	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*
	MIR+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	MIR+COC	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**

p <0.01: significant effect of cocaine treatment compared with the SAL + SAL and MIR + SAL groups. **p <0.01: significant effect of mirtazapine treatment compared with the SAL + COC group. #p <0.01: significant effect of mirtazapine treatment compared with the MIR + SAL and MIR + COC groups. +p <0.01: significant effect of mirtazapine treatment compared with the MIR + SAL and SAL + SAL groups.

In addition, our statistical analysis identified differences in entries and time spent in the EPM, the OFT, and the LDT shown by the SAL + COC group at 60 days, compared to days 1 (p < 0.002), 15 (p < 0.003) and 30 (p < 0.002) of cocaine withdrawal, as well as differences between day 15 and 30 (p < 0.003) of cocaine withdrawal.

In contrast, the post hoc analysis found that the MIR + COC group had a gradual increase in time spent and entries into the EPM (open and closed arms), the OFT (center and periphery) and the LDT (light and dark compartment) at 15 (p < 0.001), 30 (p < 0.001) and 60 (p < 0.001) days of cocaine withdrawal, compared with the SAL + COC group. However, the analysis found no differences in the EPM, the OFT and the LDT on the first day (p = 0.85) of drug withdrawal between the MIR + COC and SAL + COC groups (Table 2).

Additionally, the Tukey’s test revealed significant differences in entries and time spent in the EPM, the OFT and the LDT shown by the MIR + COC group at 60 days compared with days 1 (p < 0.001), 15 (p < 0.002) and 30 (p < 0.002) of cocaine withdrawal (Figure 2(b) to (d)).

With respect to the animals of the MIR + SAL group the statistical analysis showed differences in entries and time spent in the EPM (open and closed arms), the OFT (center and periphery) and the LDT (light and dark compartment) on the first day of drug withdrawal (p < 0.002), compared with the MIR + COC group (Table 2). Nevertheless, the post hoc test did not find differences at 15 (p = 0.95), 30 (p = 0.89) and 60 (p = 0.98) days of cocaine withdrawal between the MIR + SAL and the MIR + COC groups (Figure 2(b) to (d)).

On the other hand, the post hoc test found differences in the EPM, the OFT and the LDT at 15 (p < 0.002), 30 (p < 0.001) and 60 (p < 0.001) days of drug withdrawal between the MIR + SAL and SAL + SAL groups, but found no differences on day 1 (p = 0.78) of drug withdrawal between these groups (Figure 2(b) to (d)). In addition, the statistical analysis found differences in entries and time spent in the EPM test, the OFT and the LDT at day 60 compared with days 1 (p < 0.001), 15 (p < 0.002) and 30 (p < 0.003) of drug withdrawal in the MIR + SAL group.

Depression-like behavior

Three-way repeated measures ANOVA found differences (Table 1) between the different factors. The post hoc test found a significant increase in the immobility shown by the SAL + COC group compared with those in the SAL + SAL and the MIR + SAL groups at 1 (p < 0.001), 15 (p < 0.001), 30 (p < 0.001) and 60 (p < 0.001) days of cocaine withdrawal. In addition, the statistical analysis revealed differences in the immobility shown by the SAL + COC group at 60 days compared with 1 (p < 0.001), 15 (p < 0.001) and 30 (p < 0.003) days of drug withdrawal.

In contrast, the post hoc test found a decrease in the immobility time shown by the MIR + COC group compared with the SAL + COC and the SAL + SAL groups at 1 (p < 0.001), 15 (p < 0.001), 30 (p < 0.001) and 60 (p < 0.001) days of cocaine withdrawal. In addition, the statistical analysis identified differences in immobility shown by the MIR + COC group at 60 days compared with 1 (p < 0.001), 15 (p < 0.001) and 30 (p < 0.001) days of cocaine withdrawal (Figure 1(e)).

On the other hand, the post hoc test found differences in the immobilization time shown by the MIR + SAL group compared with the animals of the SAL + SAL group at 15 (p < 0.001), 30 (p < 0.001) and 60 (p < 0.001) days of drug withdrawal, but it did not find any differences on the first day (p = 0.95).

In contrast, the post hoc test found differences in immobilization time (p < 0.001) shown by the MIR + SAL group compared with the MIR + COC group, 15 days after the last administration of cocaine, whereas Tukey’s test did not find any differences between these groups at 1 (p = 0.98), 30 (p = 0.92) and 60 (p = 0.96) days after drug withdrawal (Figure 1(e)).

Spontaneous locomotor activity

A three-way repeated measures ANOVA found significant differences (Table 1) between the different factors. Tukey’s test found differences in SLA in the SAL + COC group compared with the SAL + SAL, the MIR + SAL, and the MIR + COC groups at 15 (p < 0.002), 30 (p < 0.001) and 60 (p < 0.001) days of drug withdrawal (Figure 1(f)). Further, our statistical analysis found differences between the MIR + SAL and the MIR + COC groups at 1 (p < 0.003), 15 (p < 0.003) and 60 (p < 0.001) days of cocaine withdrawal.

Experiment 2: Mirtazapine in different doses modified anxiety- and depression-like behaviors during cocaine withdrawal

Locomotor activity induction

Three-way ANOVA found differences (groups by treatment interaction, F (1.78) = 156.25; p < 0.0002) in the cocaine-induced locomotor activity shown by the SAL + COC, MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC groups compared with the SAL + SAL (p <0.001) and MIR + SAL (p <0.001) groups (Figure 1(b)).

Anxiety-like behavior

Four-way repeated measures ANOVA revealed significant differences between the different factors in the EPM (Figure 3(b); Table 3), the OFT (Figure 3(c); Table 3) and the LDT (Figure 3(d); Table 3) shown by the SAL + COC group at days 1 (p < 0.003), 15 (p < 0.001) and 30 (p < 0.001) of drug withdrawal (Table 4), as was the case in Experiment 1 (Figure 3(b) to (d)).

Table 3.

Comparisons between the groups, treatments, mirtazapine doses and cocaine withdrawal time by means of a four-way ANOVA during the cocaine withdrawal in the behavioral parameters evaluated with the EPM, OFT, LDT, FST and LSA.

Experiment 2	EPM				OFT				LDT				FST	SLA
Four-way ANOVA	OAEN	OAPT	CAEN	CAPT	CEN	CPT	PPT	RRG	LEN	LPT	DEN	DPT	IT	LAC
(A) Groups F (1, 168) =	74.12,p < 0.002	107.21,p < 0.002	72.94,p < 0.001	73.62,p < 0.002	49.73,p < 0.002	33.21,p < 0.004	64.51,p < 0.005	68.42,p < 0.006	46.78,p < 0.003	91.28,p < 0.002	32.81,p < 0.001	157.21,p < 0.002	77.28,p < 0.002	107.59,p < 0.001
(B) Treatments F (1, 168) =	100.44,p < 0.003	85.14,p < 0.002	66.31,p < 0.002	59.39,p < 0.002	76.98,p < 0.003	100.28,p < 0.002	75.14,p < 0.004	111.35,p < 0.003	81.35,p < 0.004	112.42,p < 0.001	124.99,p < 0.002	57.46,p < 0.002	31.78,p < 0.002	116.89,p < 0.002
(C) Treatment doses F (2, 168) =	72.33,p < 0.002	51.77,p < 0.002	82.60,p < 0.001	71.44,p < 0.003	117.51,p < 0.002	39.75,p < 0.003	126.78,p < 0.002	129.49,p < 0.002	128.39,p < 0.003	144.89,p < 0.002	47.69,p < 0.04	34.50,p < 0.02	46.58,p < 0.002	123.81,p < 0.003
(D) Cocaine withdrawal time F (2, 168) =	56.17,p < 0.003	45.21,p < 0.003	97.23,p < 0.002	98.39,p < 0.001	78.91,p < 0.004	38.69,p < 0.004	53.34,p < 0.002	80.08,p < 0.003	76.74,p < 0.003	61.89,p < 0.001	117.42,p < 0.002	125.10,p < 0.002	37.55,p < 0.002	207.14,p < 0.002
A × B × C × D interactions F (2, 168) =	95.58,p < 0.002	80.43,p < 0.004	88.77,p < 0.002	97.96,p < 0.002	49.31,p < 0.002	149.11,p < 0.002	123.29,p < 0.001	110.97,p < 0.001	25.33,p < 0.002	86.54,p < 0.002	13.07,p < 0.02	94.72,p < 0.001	19.11,p < 0.003	215.61,p < 0.001

Table 4.

Comparisons between the behavioral parameters evaluated with EPM, OFT, LDT, FST and LSA by means of a Tukey test during the 1, 15 and 30 days of cocaine withdrawal.

Experiment 2		p <	EPM			OFT			LDT
Time	Doses Mirtazapine	Group	OAPT	CAEN	CAPT	CPT	PPT	RRG	LPT	DEN	DPT
1 Day		SAL+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
		SAL+COC	0.003*	NS	NS	0.003*	0.002*	NS	0.003*	0.03*	0.03*
	15 mg/kg	MIR_-15+SAL	0.001 ^#	0.004 ^#	0.003 ^#	0.001 ^#	0.002 ^#	0.003 ^#	0.001 ^#	0.002 ^#	0.002 ^#
	30 mg/kg	MIR_-30+SAL	0.001 ^#	0.002 ^#	0.001 ^#	0.001 ^#	0.003 ^#	0.001 ^#	0.001 ^#	0.001 ^#	0.001 ^#
	60 mg/kg	MIR_-60+SAL	0.001 ^#	0.001 ^#	0.001 ^#	0.001 ^#	0.002 ^#	0.001 ^#	0.001 ^#	0.001 ^#	0.001 ^#
	15 mg/kg	MIR_-15+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
	30 mg/kg	MIR_-30+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
	60 mg/kg	MIR_-60+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
15 Day		SAL+SAL	0.004 ⁺	0.003 ⁺	0.004 ⁺	0.004 ⁺	0.002 ⁺	0.002 ⁺	0.004 ⁺	0.002 ⁺	0.003 ⁺
		SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.003*
	15 mg/kg	MIR_-15+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	30 mg/kg	MIR_-30+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	60 mg/kg	MIR_-60+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	15 mg/kg	MIR_-15+COC	0.001**	0.002**	0.001**	0.001**	0.003**	0.003**	0.001**	0.002**	0.001**
	30 mg/kg	MIR_-30+COC	0.001**	0.001**	0.002**	0.001**	0.001**	0.001**	0.001**	0.002**	0.002**
	60 mg/kg	MIR_-60+COC	0.001**	0.003**	0.002**	0.001**	0.001**	0.002**	0.001**	0.001**	0.001**
30 Day		SAL+SAL	0.003 ⁺	0.003 ⁺	0.002 ⁺	0.003 ⁺	0.001 ⁺	0.001 ⁺	0.003 ⁺	0.003 ⁺	0.003 ⁺
		SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.002*	0.002*
	15 mg/kg	MIR_-15+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	30 mg/kg	MIR_-30+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	60 mg/kg	MIR_-60+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	15 mg/kg	MIR_-15+COC	0.001**	0.002**	0.003**	0.001**	0.002**	0.002**	0.001**	0.001**	0.001**
	30 mg/kg	MIR_-30+COC	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**
	60 mg/kg	MIR_-60+COC	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**	0.001**

p <0.01: significant effect of cocaine treatment compared with the SAL + SAL and MIR + SAL groups. **p <0.01: significant effect of mirtazapine treatment compared with the SAL + COC group. #p <0.01: significant effect of mirtazapine treatment compared with the MIR + SAL and MIR + COC groups. +p <0.01: significant effect of the mirtazapine treatment compared with the MIR + SAL and SAL + SAL groups.

In contrast, the administration of mirtazapine to the animals in the MIR-_15mg + COC, the MIR-_30mg + COC and the MIR-_60mg + COC groups produced a gradual increase in entries and time spent in the EPM (open and closed arms; Figure 3(b)), the OFT (center and periphery; Figure 2(c)) and the LDT (light and dark compartment; Figure 3(d)) compared with the SAL + COC group at 15 (p < 0.001) and 30 days (p < 0.001) after drug withdrawal. Further, on the first day of cocaine withdrawal, the statistical analysis found no differences (p = 0.94) between the MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC groups and the SAL + COC group.

In addition, Tukey’s test revealed differences in the EPM, the OFT and the LDT test between the animals dosed with 15 mg mirtazapine (MIR-_15mg + COC) compared with the MIR-_30mg + COC and the MIR-_60mg + COC groups at 15 (p < 0.003) and 30 (p < 0.002) days after drug withdrawal, whereas the statistical analysis found no difference (p = 0.88) between the MIR-_30mg + COC and the MIR-_60mg + COC groups during drug withdrawal (Table 4).

With the post hoc test, we compared the MIR-_15mg + SAL, MIR-_30mg + SAL and MIR-_60mg + SAL groups to the SAL + SAL group and found differences in entries and time spent in the EPM (open and closed arms), the OFT (center and periphery) and the LDT (light and dark compartment) at 15 (p < 0.004) and 30 days (p < 0.003) of drug withdrawal. Nevertheless, we did not find differences (p = 0.89) on the first day of cocaine withdrawal (Figure 3(b) to (d)).

The statistical analysis also found differences on day 1 after cocaine withdrawal in the EPM, the OFT and the LDT shown by the MIR-_15mg + SAL, the MIR-_30mg + SAL and the MIR-_60mg + SAL groups compared with the MIR-_15mg + COC (p < 0.001), the MIR-_30mg + COC (p < 0.001) and the MIR-_60mg + COC (p < 0.001) groups. The post hoc test, however, did not indicate differences after 15 (p = 0.76) and 30 (p = 0.87) days of drug withdrawal (Table 4; Figure 3(b) to (d)).

Depression-like behaviors

With regard to the FST, the four-way repeated measures ANOVA revealed significant differences (Table 3) between the different factors. With Tukey’s test, we found that the SAL + COC group had an increase in immobility time compared with the SAL + SAL group at 15 (p < 0.001) and 30 (p < 0.001) days of drug withdrawal, whereas the post hoc test did not show any differences (p = 0.97) in immobility time on the first day of cocaine withdrawal (Figure 3(e)).

The animals in the MIR-_15mg + COC, the MIR-_30mg + COC, and the MIR-_60mg + COC groups had a gradual decrease in immobility. The post hoc test found differences in immobility time at 15 (p < 0.001) and 30 (p < 0.001) days of cocaine withdrawal when comparing the MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC groups to the SAL + COC group. Nonetheless, a day after drug withdrawal, Tukey’s test did not find (p = 0.94) any differences in immobility in the MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC groups compared to the SAL + COC group. The statistical analysis identified differences in immobility time in the MIR-_15mg + COC group compared with the MIR-_30mg + COC and the MIR-_60mg + COC groups at 15 (p < 0.003) and 30 (p < 0.003) days of drug withdrawal (Figure 3(e)).

When the immobility showed by the MIR-_15mg + SAL, MIR-_30mg + SAL and MIR-_60mg + SAL groups was compared with that shown by the SAL + SAL group, a decrease was found at 15 (p < 0.001) and 30 (p < 0.001) days of drug withdrawal. On the other hand, when comparing the MIR-_15mg + SAL, MIR-_30mg + SAL and MIR-_60mg + SAL groups to the MIR-_15mg + COC, MIR-_30mg + COC and MIR-_60mg + COC groups, the statistical analysis did not find differences in immobility time at 15 (p = 0.89) and 30 (p = 0.92) days of cocaine withdrawal. But, it did find differences (p < 0.004) on the first day of drug withdrawal among these groups (Figure 3E).

Spontaneous locomotor activity

The four-way repeated measures ANOVA revealed significant differences (Table 3) between the different factors. Tukey’s test found differences in SLA in the animals in the SAL + COC group compared with those in the SAL + SAL, MIR-_15mg + SAL, MIR-_30mg + SAL, MIR-_60mg + SAL, MIR-_15mg + COC, MIR-_30mg + COC, and MIR-_60mg + COC groups at days 1 (p < 0.001), 15 (p < 0.001) and 30 (p < 0.001) of drug withdrawal (Figure 3(f)). The statistical analysis found no differences (p = 0.76) between the MIR-_15mg + SAL, MIR-_30mg + SAL and MIR-_60mg + SAL groups at the above-mentioned days. Additionally, the post hoc test showed differences in SLA in the MIR-_15mg + COC group compared with the MIR-_30mg + COC and MIR-_60mg + COC groups at days 15 (p < 0.002) and 30 (p < 0.003) of cocaine withdrawal.

Experiment 3: Mirtazapine modified the anxiety- and depression-like behaviors

Locomotor activity induction

The statistical analysis (three-way ANOVA; in the groups by treatment interaction, F (1. 88) = 81.28; p < 0.0001) found that during the induction phase different doses of cocaine increased the locomotor activity in the SAL + COC-_10mg, SAL + COC-_20mg, SAL + COC-_40mg, MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups compared with the SAL + SAL (p <0.001) and MIR + SAL (p <0.001) groups (Figure 1(c)).

Anxiety-like behaviors

For the EPM (Table 5; Figure 4(b)), the OFT (Table 5; Figure 4(c)), and the LDT (Table 5; Figure 4(d)), the four-way repeated measures ANOVA revealed significant differences between the different factors. The post hoc test found a dose-dependent decrease in entries and time spent in the open and closed arms, the center and periphery of the open field, and the light and dark compartment shown by the SAL + COC-_10mg, SAL + COC-_20mg, and SAL + COC-_40mg groups compared with the SAL + SAL and MIR + SAL groups at days 1 (p < 0.001), 15 (p < 0.001) and 30 (p < 0.001) after cocaine withdrawal (Table 6).

Table 5.

Comparisons between the groups, treatments cocaine doses and cocaine withdrawal time by means of a four-way ANOVA during cocaine withdrawal in the behavioral parameters evaluated with EPM, OFT, LDT, FST and LSA.

Experiment 3	EPM				OFT				LDT				FST	SLA
Four-way ANOVA	OAEN	OAPT	CAEN	CAPT	CEN	CPT	PPT	RRG	LEN	LPT	DEN	DPT	IT	LAC
(A) Groups F (1, 148) =	50.28,p < 0.001	111.73,p < 0.002	70.35,p < 0.001	75.83,p < 0.001	139.82,p < 0.001	129.46,p < 0.003	77.03,p < 0.004	113.98,p < 0.004	46.80,p < 0.002	73.84,p < 0.002	78.23,p < 0.003	34.97,p < 0.002	18.23,p < 0.004	248.71,p < 0.001
(B) Treatments F (1, 148) =	44.89,p < 0.002	102.37,p < 0.002	92.77,p < 0.001	108.18,p < 0.003	173.85,p < 0.004	228.94,p < 0.002	127.93,p < 0.002	93.67,p < 0.003	51.62,p < 0.003	39.41,p < 0.001	79.66,p < 0.001	81.74,p < 0.001	34.82,p < 0.002	103.49,p < 0.002
(C) Doses treatments F (2,148) =	115.29,p < 0.003	172.91,p < 0.002	88.05,p < 0.002	112.98,p < 0.002	186.48,p < 0.001	271.59,p < 0.001	171.08,p < 0.003	95.93,p < 0.002	73.84,p < 0.003	69.90,p < 0.003	41.26,p < 0.002	53.47,p < 0.004	27.52,p < 0.001	130.23,p < 0.001
(D) Cocaine withdrawal time F (2, 148) =	46.18,p < 0.004	74.21,p < 0.002	97.23,p < 0.02	50.88,p < 0.001	121.38,p < 0.003	289.18,p < 0.002	77.91,p < 0.04	94.74,p < 0.003	53.37,p < 0.001	26.87,p < 0.002	97.36,p < 0.005	22.36,p < 0.002	48.58,p < 0.004	182.13,p < 0.001
A × B × C × D Interactions. F (2, 148) =	15.12,p < 0.003	22.73,p < 0.003	21.15,p < 0.001	39.72,p < 0.002	52.12,p < 0.002	44.52,p < 0.003	46.28,p < 0.002	60.05,p < 0.002	95.13,p < 0.001	317.72,p < 0.001	88.85,p < 0.003	98.87,p < 0.002	39.02,p < 0.003	144.78,p < 0.001

Table 6.

Comparisons between the groups the behavioral parameters evaluated with the EPM, OFT, LDT, FST and LSA by means of a Tukey test during the 1, 15, and 30 days of cocaine withdrawal.

Experiment 3			EPM			OFT			LDT
Time	Doses of cocaine	Group	OAPT	CAEN	CAPT	CPT	PPT	RRG	LPT	DEN	DPT
1 day		SAL+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	10 mg/kg	SAL+COC	0.001*	NS	NS	0.001*	0.003*	NS	0.001*	0.002*	0.002*
	20 mg/kg	SAL+COC	0.001*	0.002*	0.001*	0.001*	0.002*	0.002*	0.001*	0.001*	0.001*
	40 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*
		MIR+SAL	0.003 ^#	0.002 ^#	0.004 ^#	0.003 ^#	0.003 ^#	0.001 ^#	0.003 ^#	0.002 ^#	0.002 ^#
	10 mg/kg	MIR+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
	20 mg/kg	MIR+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
	40 mg/kg	MIR+COC	NS	NS	NS	NS	NS	NS	NS	NS	NS
15 days		SAL+SAL	0.001 ⁺	0.003 ⁺	0.003 ⁺	0.001 ⁺	0.002 ⁺	0.002 ⁺	0.001 ⁺	0.002 ⁺	0.002 ⁺
	10 mg/kg	SAL+COC	0.001*	0.003*	0.002*	0.001*	0.001*	0.002*	0.001*	0.001*	0.001*
	20 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*
	40 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*
		MIR+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	10 mg/kg	MIR+COC	0.001**	0.002**	0.002**	0.001**	0.003**	0.002**	0.001**	0.002**	0.002**
	20 mg/kg	MIR+COC	0.001**	0.001**	0.002**	0.002**	0.002**	0.001**	0.001**	0.002**	0.001**
	40 mg/kg	MIR+COC	0.001**	0.001**	0.001**	0.002**	0.003**	0.001**	0.001**	0.001**	0.002**
30 days		SAL+SAL	0.001 ⁺	0.003 ⁺	0.003 ⁺	0.002 ⁺	0.002 ⁺	0.002 ⁺	0.001 ⁺	0.002 ⁺	0.003 ⁺
	10 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.002*	0.002*	0.001*	0.001*	0.001*
	20 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.002*	0.001*	0.001*	0.001*	0.001*
	40 mg/kg	SAL+COC	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*	0.001*
		MIR+SAL	NS	NS	NS	NS	NS	NS	NS	NS	NS
	10 mg/kg	MIR+COC	0.001**	0.001**	0.001**	0.001**	0.002**	0.001**	0.001**	0.001**	0.001**
	20 mg/kg	MIR+COC	0.001**	0.001**	0.002**	0.001**	0.002**	0.003**	0.001**	0.002**	0.003**
	40 mg/kg	MIR+COC	0.001**	0.001**	0.002**	0.001**	0.003**	0.002**	0.001**	0.002**	0.002**

p <0.01: significant effect of cocaine treatment compared with the SAL + SAL and MIR + SAL groups. **p <0.01: significant effect of mirtazapine treatment compared with the SAL + COC group. #p <0.01: significant effect of the mirtazapine treatment compared with the MIR + SAL and MIR + COC groups. +p <0.01: significant effect of the mirtazapine treatment compared with the MIR + SAL and SAL + SAL groups.

Further, with the post hoc test, we found differences in the EPM, the OFT and the LDT in the group treated with 40 mg cocaine (SAL + COC-_40mg) compared with the SAL + COC-_10mg (p < 0.001) and the SAL + COC-_20mg (p < 0.001) groups during cocaine withdrawal. The post hoc test revealed differences between the SAL + COC-_10mg and the SAL + COC-_20mg (p < 0.003) groups (Table 6). There were also differences in entries and time spent in the EPM, the OFT and the LDT between the SAL + COC-_10mg, the SAL + COC-_20mg, and the SAL + COC-_40mg groups at days 1 (p < 0.001), 15 (p < 0.003) and 30 (p < 0.003) of drug withdrawal (Figure 4(b) to (d)).

Mirtazapine administered to the animals in the MIR + COC-_10mg, the MIR + COC-_20mg and the MIR + COC-_40mg groups resulted in a gradual, cocaine-dose independent increase in entries and time spent in the EPM (open and closed arms), the OFT (center and periphery) and the LDT (light and dark compartment) at days 15 (p < 0.001) and 30 (p < 0.001) after cocaine withdrawal, compared with the SAL + COC-_10mg, the SAL + COC-_20mg and the SAL + COC-_40mg groups.

By comparing the MIR + COC-_40mg group to the MIR + COC-_10mg and MIR + COC-_20mg groups, Tukey’s test found differences in the EPM, the OFT, and the LDT at days 15 (p < 0.001) and 30 (p < 0.001) after cocaine withdrawal, but none (p = 0.92) on the first day. Tukey’s test also identified differences between the MIR + COC-_10mg and the MIR + COC-_20mg (p < 0.003) groups (Table 6).

The post hoc test found differences between the MIR + SAL group and the SAL + SAL group in entries and time spent in the EPM (open and closed arms), the OFT (center and periphery) and the LDT (light and dark compartment) at 15 (p < 0.001) and 30 (p < 0.001) days but none (p = 0.89) at day 1 (Figure 4(b) to (d)).

In addition, our statistical analysis found differences in the EPM, the OFT and the LDT shown by the MIR + SAL group compared with the MIR + COC-_10mg (p < 0.003) and the MIR + COC-_20mg (p < 0.003) groups at day 1 of cocaine withdrawal, but the post hoc test did not find differences at days 15 (p = 0.88) and 30 (p = 0.78) (Table 6). Nevertheless, the statistical analysis showed differences in the EPN, the OFT and the LDT in the MIR + COC-_40mg group compared with the MIR + SAL group (p < 0.001).

Depression-like behaviors

Figure 4(e) shows that the animals in the SAL + COC-_10mg, the SAL + COC-_20mg and the SAL + COC-_40mg groups showed a significant increase in immobility time during cocaine withdrawal (Table 5). Further, after the last cocaine injection, the post hoc test revealed that compared with the SAL + SAL and MIR + SAL groups, the SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg groups had a significant cocaine dose-independent increase in immobility at days 15 (p < 0.001) and 30 (p < 0.001) but none at day 1 (p = 0.91; Figure 4(e)).

In contrast, the rats in the MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups showed a gradual decrease in immobility time, compared with those in the SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg groups. The post hoc test did not find differences (p = 88) between the groups that received the different doses of cocaine in both treatments (SAL or MIR) on the first day of drug withdrawal. However, at 15 (p < 0.001) and 30 (p < 0.001) days of cocaine withdrawal, the post hoc test found differences in the immobility shown by the SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg groups compared with that shown by the MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups (Figure 4(e)).

In addition, the post hoc test found differences in immobility time shown by the MIR + SAL group compared with those in the SAL + SAL group at 15 (p < 0.003) and 30 (p < 0.002) days of drug withdrawal. Immobility in the MIR + SAL group compared with that in the SAL + SAL group did not differ on the first day of drug withdrawal (p = 0.94).

Similarly, the post hoc test found differences in immobility time (p < 0.003) when comparing the MIR + SAL to the MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups 1 day after the last cocaine administration. Nonetheless, at 15 (p = 0.73) and 30 (p = 0.80) days of drug withdrawal, Tukey’s test did not reveal any differences (Figure 4(e)).

Spontaneous locomotor activity

The four-way repeated measures ANOVA revealed significant differences between the different factors (Table 5). Tukey’s test found differences in SLA in the SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg groups compared with the SAL + SAL, MIR + SAL, MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups at 15 (p < 0.001) and 30 (p < 0.001) days of cocaine withdrawal (Figure 4(f)). The statistical analysis found differences in locomotor activity between the SAL + COC-_10mg, SAL + COC-_20mg and SAL + COC-_40mg groups at days 1 (p < 0.001), 15 (p < 0.003) and 30 (p < 0.003) of withdrawal. In addition, Tukey’s test found no differences between the MIR + SAL and the MIR + COC-_10mg, MIR + COC-_20mg and MIR + COC-_40mg groups at days 15 (p = 0.88) and 30 (p = 0.88) of drug withdrawal.

Discussion

Several clinical trials have reported that discontinuation of drug intake in cocaine abusers usually produces a variety of adverse withdrawal symptoms, including sleep disturbances, anxiety- and depression-like behaviors (El-Hage et al., 2012; Lai et al., 2015). Similar results have been reported in studies of rodents, which showed an increase in depression- and anxiety-like behaviors when exposed to different periods of drug withdrawal (Paine et al., 2002; Perrine et al., 2008; Rudoy and Van-Bockstaele, 2007).

These studies have also shown that cocaine withdrawal results in an intense depression- and anxiety-like behavior characterized by a decrease in exploration in the EPM and OFT, as well as an increase in immobility in FST, from the first day of abstinence (Erb et al., 2006; Sarnyai et al., 1995). We found similar results. From day 1 of cocaine withdrawal, we noted a gradual decrease in entries and time spent in the EPM (open arms), the OFT (center) and the LDT (light compartment), as well as an increase in immobility in the FST.

We also found that depression- and anxiety-like behaviors increased throughout drug withdrawal. Our statistical analysis found differences in such behaviors at 60 days, compared with those observed at 15 and 30 days of cocaine withdrawal.

These observations are consistent with reports that indicate that depression- and anxiety-like behaviors persist for up to 28 days (El-Hage et al., 2012), but they differ from other clinical and preclinical reports that show that states of anxiety and depression decrease with long-term abstinence (Gawin and Kleber, 1986; Stoker and Markou, 2011). Our results suggest that during a long period of abstinence the symptoms of anxiety and depression increase.

Other studies have reported that locomotor activity decreases during cocaine withdrawal (Calipari et al., 2013). Our findings point toward a different conclusion: compared with baseline values, SLA increased, which suggests that the rats may have been in a state of anxiety.

Experiment 1: Mirtazapine reduced anxiety- and depression-like behaviors during cocaine withdrawal

Our study found that dosing of 30 mg/kg mirtazapine decreased anxiety, as measured in the EPM, OFT and LDT, as well as depression-like behaviors in the FST in animals previously treated with 10 mg/kg cocaine at 15, 30 and 60 days of drug withdrawal. These results agree with clinical trials in patients with a comorbid major depressive disorder and alcohol or cocaine dependence, where mirtazapine significantly improves symptoms of depression, anxiety and insomnia, while minimizing the physical and subjective discomfort and dysphoric symptoms of benzodiazepine, alcohol and cocaine withdrawal (Afshar et al., 2012; Chandrasekaran, 2008; Cornelius et al., 2012, 2013, 2016).

Previous reports have indicated that treatment with antidepressants such as fluoxetine, venlafaxine and reboxetine (Kampman et al., 2001a, 2001b; Lapmanee et al., 2012, 2013; Rénéric et al., 2002; Rogóż and Kabziński, 2011a; Rogóż and Skuza, 2011b) decrease anxiety-like behavior in the EPM (Drapier et al., 2007; Silva et al., 1999) and depression-like behaviors in the FST on the first day of drug withdrawal (Muguruza et al., 2013), but these effects decrease at day 28 (Nowakowska, et al., 1996; Robert et al., 2011). Our results differ from these studies in that dosing of mirtazapine did not reduce anxiety- and depression-like behaviors on the first day of treatment. Nevertheless, 15 days after cocaine withdrawal, mirtazapine exerted noticeable and persistent anxiolytic and antidepressant effects. These results are consistent with previous reports from our laboratory that show that dosing of mirtazapine for 60 days leads to an improvement in the effects on cocaine-induced locomotor activity observed after 30 days (Barbosa-Méndez et al., 2017b).

It has also been reported that treatment with ondansetron, a 5-HT₃ receptor antagonist (de Oliveira Citó Mdo et al., 2012), or buspirone, a 5-HT₁ receptor agonist, decreases depression- and anxiety-like behaviors at 24 hours and 7 days of abstinence (Kolcsar et al., 2014). This suggests, on the one hand, the involvement of the serotonergic system in the genesis of anxiety and depression symptoms; on the other hand, it suggests that mirtazapine – through its simultaneous pharmacological effects on 5-HT₁, 5-HT₂ and 5-HT₃ receptors – decreases long-term depression- and anxiety-like behaviors as evaluated in the EPM, OFT, LDT and FST.

Some authors have also found that during drug withdrawal there are deregulated levels of catecholamines (DA and NE), which leads to the development of anxiety and depression symptoms (Dworkin et al., 1995). Mirtazapine is known to produce its therapeutic antidepressant and anxiolytic effects by increasing 5-HT and NE levels (de Boer, 1996; de Boer et al., 1996). Thus, mirtazapine-induced restoration of levels of brain catecholamines may be part of a neurobiological mechanism through which mirtazapine steadily decreases anxiety- and depression-like behaviors during cocaine withdrawal.

Several research studies have suggested that mirtazapine decreases anxiety-like behavior in the EPM and depression-like behaviors in the FST in animals treated with saline (An et al., 2013, 2015, 2016a, 2016b; Kaminska and Rogoz, 2016; Rogóż, 2010, 2012; Rogóż et al., 2012). We found similar results: in animals dosed with 30 mg mirtazapine, anxiety-like (as measured in the EPM test, OFT and the LDT) and depression-like behaviors (according to the FST) decreased after 15 days of treatment. Studies elsewhere have indicated that mirtazapine exerts rapid antidepressant effects compared with drugs such as the SSRIs (Hashimoto et al., 2016; Tsutsumi et al., 2016). Again, our results were similar – after only 15 days of treatment, we noted a decrease in depression- and anxiety-like behaviors. Collectively, these results support the assumption that mirtazapine has therapeutic (anxiolytic and antidepressant) properties.

However, one limitation of this study is the use of male rats to evaluate the effect of mirtazapine on depression- and anxiety-like behaviors during cocaine withdrawal. During drug withdrawal, women exhibit more intense withdrawal symptoms and cocaine-craving than men, which makes women more vulnerable than men in terms of treatment outcomes (Becker, 2016).

In animals, the results are similar; females rats are more sensitive to the reinforcing and behavioral-activating effects of cocaine and exhibit an increase in vulnerability during the acquisition and relapse/reinstatement phases (Anker and Carroll, 2010; Fuchs et al., 2005).

Therefore, future studies are required to determine the efficacy of mirtazapine in terms of reducing depression- and anxiety-like behaviors during cocaine withdrawal in female rats.

Experiment 2: Mirtazapine in different doses modified anxiety- and depression-like behaviors during cocaine withdrawal

Previous studies from our laboratory reported that 60 mg/kg mirtazapine does not improve the decrease in cocaine-induced locomotor activity produced by 30 mg/kg of mirtazapine (Barbosa-Méndez et al., 2017b). Additionally, it has been described that the increase in the dose of mirtazapine beyond 30 mg/day does not increase the effectiveness of mirtazapine in patients with depression (Ueno et al., 2015).

In this study, we found similar results. A dose of 60 mg/kg of mirtazapine did not improve the reduction of depression- and anxiety-like behaviors compared with those produced by a dose of 30 mg/kg. This effect was not due to a decrease in SLA, since in this study the dosage of 60 mg/kg of mirtazapine did not decrease the SLA.

We have also reported that a dose of 15 mg/kg did not decrease cocaine-induced locomotor activity (Barbosa-Méndez et al., 2017b). In this study, 15 mg/kg mirtazapine decreased anxiety-like behavior in the EPM, OFT and LDT, as well as depression-like behavior in the FST, 15 days after cocaine withdrawal. Clinical studies have suggested that 15 mg/kg mirtazapine successfully reduces depression and insomnia in patients with major depressive disorder (Grasmäder et al., 2005; Kamphuis et al., 2015; Matreja et al., 2012). Other authors have shown that even doses ⩽ 10 mg/kg reduce both anxiety-like behavior in the EPM and depression-like behaviors in the FST in rats treated with saline (Kaminska and Rogoz, 2016; Rogóż, 2012).

Clinical trials have reported that an increase in the dosage of antidepressants such as fluoxetine (Jakubovski et al., 2016; Venkatasubramanian et al., 2013) may be needed in order to treat chronic episodes of depression. It has also been found that higher antidepressant doses produce increased anxiolytic (in the EPM) and antidepressant (in the FST) effects in rats (Hodes et al., 2010; Kolcsar et al., 2014). Still, in both cases, the increase in dose produces serious adverse effects (Hodes et al., 2010; Olivier et al., 2011).

Post-mortem studies of subjects with major depression suggested that one of the mechanisms of action of some medications, such as mirtazapine, is downregulation of 5-HT_2A receptor expression in the prefrontal cortex (PFC) (Muguruza et al., 2014). Other studies have reported that treatment with antidepressants does not change the expression and density of 5-HT_2C receptors (Millan, 2005), which are important in the treatment of depression. On the other hand, it has been reported that repeated mirtazapine administration does not alter the expression of the D₂/D₃ dopamine receptors (Rogóz et al., 2002a), but it increases the expression of the D₁ dopamine receptor (Huzarska et al., 2006) and 5-HT_2C receptors (Chanrion et al., 2008) and the responsiveness of the α₁-noradrenergic receptors (Rogóż et al., 2002b), which suggests that mirtazapine can differentially affect certain receptor populations in the brain.

However, to our knowledge there is no study that evaluates the effect of different doses of mirtazapine on the expression of 5-HT receptors in different regions of the brain. Therefore, future dose–response studies aimed at describing the effect of mirtazapine on the expression of 5-HT receptors in discrete regional populations are required.

Experiment 3: Mirtazapine modified the anxiety- and depression-like behaviors

Earlier research has demonstrated that higher dosing of cocaine enhances its reinforcing effects (Barbosa-Méndez et al., 2017b). In addition, several authors have described the effect of withdrawal of different doses of cocaine on depression- and anxiety-like behaviors (El-Hage et al., 2012; Erb et al., 2006; Stoker and Markou, 2011). Specifically, 15 and 20 mg/kg of cocaine reduced the number of entries and time spent in the open arms of the EPM. Our study yielded similar results. As cocaine doses increased, so did depression- and anxiety-like behaviors during cocaine withdrawal. In fact, we found that 40 mg/kg of cocaine increased anxiety-like behavior (EPM, OFT and LDT) and depression-like behaviors (FST). Erb et al. reported similar findings: they showed that 30 mg/kg of cocaine raises levels of anxiety (Erb et al., 2006).

Another finding of our study was that 30 mg/kg mirtazapine decreased depression- and anxiety-like behaviors induced by withdrawal of 10 and 20 mg/kg of cocaine. Our mirtazapine dosing schedule (30 mg/kg for 30 days) was enough to decrease anxiety-like behavior in the EPM, OFT and LDT and depression-like behaviors in the FST during withdrawal of 40 mg/kg cocaine, which suggests that chronic dosing of mirtazapine may have restored brain levels of catecholamines. Nevertheless, further micro-dialysis studies are required to prove this hypothesis.

General discussion

Anxiety and depression are key symptoms of the cocaine withdrawal syndrome in human addicts, and some consider them the main factors that precipitate relapse to chronic cocaine abuse (Rudoy and Van-Bockstaele, 2007). These symptoms occur within 24 hours of cessation of dosing and persist throughout the period of abstinence (Walsh et al., 2009). In fact, the history of cocaine withdrawal symptoms may be associated with better cocaine responses and greater severity of cocaine dependence (Sofuoglu et al., 2003, 2005, 2006). Hence, it is important to find an effective therapy that decreases these symptoms.

In double-blind, placebo-controlled studies in patients with comorbid major depressive disorder and alcohol or cocaine dependence, mirtazapine produced a significant decrease in depressive symptoms at week 2, and at all subsequent evaluations (weeks 3, 4, 6, 8, 10 and 12) during a 12-week study (Afshar et al., 2012; Cornelius et al., 2012, 2013, 2016). Our results are consistent with those of the above research and suggest that the pharmacological effect of mirtazapine on brain neurotransmitter systems is to reduce depression- and anxiety-like behaviors – even when produced by sub-lethal doses of cocaine – for long periods.

Numerous studies have described that the response to antidepressant treatment is associated with changes in the expression of various proteins (Landgrebe et al., 2002; Slattery et al., 2005; Woo et al., 2018). Some of these studies have indicated that mirtazapine increases the mRNA and protein levels of BNDF and p-CREB in the hippocampus and PFC (Rogóż et al., 2005, 2017). Other studies describe that mirtazapine decreases the expression of some apoptotic proteins (Bcl-xL, Bax, Bad and p53) and increases the expression of neurotrophins (NGF and NT-3) in the hippocampus and PFC (Engel et al., 2013; Landgrebe et al., 2002; Woo et al., 2018). This suggests that mirtazapine could elicit its therapeutic effect by modulating the activity of apoptotic and neurotrophic pathways, thus improving plasticity and cell survival in depressive patients, in addition to its pharmacological effects on receptors 5-HT_1A (agonism) and 5-HT_2C (antagonism; de Boer, 1995, 1996). This supports the use of mirtazapine as a potentially effective therapy to reduce depression- and anxiety-like behaviors in cocaine withdrawal.

Footnotes

Authorship contributions

AS-J carried out the research design; AS-J and SBM conducted the experiments; AS-J performed the data analysis. AS-J wrote the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by grant INP-2040 and those awarded by Comision Nacional contra las Adicciones (CONADIC), the Gonzalo Ríos Arronte Foundation and Merck, Sharp & Dohme/Schering-Plough Mexico, SANFER Mexico.

ORCID iD

Alberto Salazar-Juárez

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