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Pharmacological evidence for the mediation of the panicolytic effect of fluoxetine by dorsal periaqueductal gray matter μ-opioid receptors
Neuropharmacology 99 (2015) 620e626
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Pharmacological evidence for the mediation of the panicolytic effect of fluoxetine by dorsal periaqueductal gray matter m-opioid receptors Camila Marroni Roncon a, d, Rafael Carvalho Almada a, d, Jhonatan Christian Maraschin b, lio Zangrossi Jr. c, e, Frederico Guilherme Graeff d, e, Elisabeth Aparecida Audi b, He a, d, e, * Norberto Cysne Coimbra ~o Preto Medical School of the University of Sa ~o Paulo, Av. dos Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeira ~o Preto, Sa ~o Paulo 14049-900, Brazil Bandeirantes, 3900, Ribeira , Av. Colombo, 5790, Maringa , Parana Laboratory of Psychopharmacology, Department of Pharmacology and Therapeutics, State University of Maringa 87020-900, Brazil c ~o Preto Medical School of the University of Sa ~o Paulo, Av. dos Bandeirantes, 3900, Ribeira ~o Preto, Sa ~o Paulo 14049Department of Pharmacology, Ribeira 900, Brazil d ~o Preto, Sa ~o Paulo 14050-220, Brazil Behavioural Neurosciences Institute (INeC), Av. do Caf e, 2450, Monte Alegre, Ribeira e ~o Preto School of Medicine of the University of Sa ~o Paulo (FMRP-USP), Av. dos Neurobiology of Emotions Research Centre (NAP-USP-NuPNE), Ribeira ~o Preto, Sa ~o Paulo 14049-900, Brazil Bandeirantes, 3900, Ribeira a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Received in revised form 20 August 2015 Accepted 21 August 2015 Available online 28 August 2015
Previously reported results have shown that the inhibitory effect of fluoxetine on escape behavior, interpreted as a panicolytic-like effect, is blocked by pretreatment with either the opioid receptor antagonist naloxone or the 5-HT1A receptor (5-HT1A-R) antagonist WAY100635 via injection into the dorsal periaqueductal gray matter (dPAG). Additionally, reported evidence indicates that the m-opioid receptor (MOR) interacts with the 5-HT1A-R in the dPAG. In the present work, pretreatment of the dPAG with the selective MOR blocker CTOP antagonized the anti-escape effect of chronic fluoxetine (10 mg/kg, i.p., daily, for 21 days), as measured in the elevated T-maze (ETM) test, indicating mediation of this effect by the MOR. In addition, the combined administration of sub-effective doses of the selective MOR agonist DAMGO (intra-dPAG) and sub-effective doses of chronic as well as subchronic (7 days) fluoxetine increased avoidance and escape latencies, suggesting that the activation of MORs may facilitate and accelerate the effects of fluoxetine. The current observation that MORs located in the dPAG mediate the anti-escape effect of fluoxetine may open new perspectives for the development of more efficient and fast-acting panic-alleviating drugs. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Dorsal periaqueductal gray matter m-opioid receptor 5-HT1A receptor Fluoxetine Elevated T-maze Panic
1. Introduction A wealth of reported evidence supports a key role of the dorsal periaqueductal gray matter (dPAG) in the regulation of both behavioral and neurovegetative manifestations of defensive reactions (Graeff, 1990, 1994; Bandler and Shipley, 1994; Lovick, 2000; Eichenberger et al., 2002; Ribeiro et al., 2005; Coimbra
Abbreviations: 5-HT1A-R, 5-HT1A receptor; dPAG, dorsal periaqueductal gray matter; MOR, m-opioid receptor; ETM, elevated T maze; 5-HT, serotonin. * Corresponding author. Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeir~ ao Preto Medical School of the University of ~o Preto, Sa ~o Paulo 14049-900, Brazil. S~ ao Paulo, Av. dos Bandeirantes, 3900, Ribeira E-mail address: [email protected] (N.C. Coimbra). http://dx.doi.org/10.1016/j.neuropharm.2015.08.037 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
et al., 2006; Twardowschy and Coimbra, 2015). Electrical or chemical stimulation of the dPAG in rodents causes behavioral changes characterized by vigorous, non-oriented escape reactions accompanied by autonomic responses, such as tachycardia, exophthalmia and increased blood pressure, which resemble those expressed in highly aversive situations such as during a predatory attack (Schutz et al., 1985; Jenck et al., 1995; Schenberg et al., 2001; Ullah et al., 2015; Almada and Coimbra, 2015; Almada et al., 2015). In humans, electrical stimulation of this structure evokes marked fear or terror, feelings of imminent death, and an urge to flee (Amano et al., 1978; Nashold et al., 1969). It has been suggested that the dPAG is fundamental for the organization of defensive behaviors related to coping with proximal threat/danger (Fanselow and Lester, 1988; Fanselow, 1991) and that malfunctioning of this
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mechanism may lead to increased susceptibility to the panic attacks that characterizes panic disorder (Deakin and Graeff, 1991; Del-Ben and Graeff, 2009; Graeff, 2004). There is substantial experimental evidence indicating that serotonin (5-HT) inhibits the escape response generated by stimulation of the dPAG (Graeff, 2004; Zanoveli et al., 2003, 2010; Graeff and Zangrossi Jr., 2010). In addition, recent studies suggest that 5HT and endogenous opioid peptides act together in the organization of defensive reactions evoked by proximal danger (Roncon et al., 2012, 2013; Rangel et al., 2014). Initially, it was shown that the selective 5-HT1A receptor (5-HT1A-R) antagonist WAY-100635 attenuated the anti-escape effect of low doses of the nonselective opioid receptor agonist morphine, suggesting a pharmacological interaction between 5-HT1A-Rs and endogenous opioids (Roncon et al., 2013). In this study, escape performance was measured in the elevated T-maze (ETM), which has been pharmacologically validated as an animal model of panic. The ETM also allows the measurement of avoidance, which has been related, in terms of psychopathology, to generalized anxiety disorder (Graeff et al, 1998; Pinheiro et al., 2007; Zangrossi Jr. et al, 2001; Moreira et al., 2013; Zangrossi Jr. and Graeff, 2014). Subsequently, it was demonstrated that the selective m-opioid receptor (MOR) antagonist CTOP attenuated the anti-escape effect of the selective 5-HT1AR agonist 8-OH-DPAT. In addition, sub-effective doses of both 8-OHDPAT and the selective MOR agonist DAMGO increased escape latencies in the elevated T-maze, which was interpreted as an antipanic effect. This set of information suggests that 5-HT1A-Rs and MORs in the dPAG interact cooperatively in the modulation of proximal defense (Roncon et al., 2013). The evidence cited above may be relevant for understanding the mechanism of action of anti-panic drugs. Thus, it has been shown that both WAY-100635 and the non-selective opioid receptor antagonist naloxone block the anti-escape effect of chronic treatment with the widely used anti-panic agent fluoxetine in the ETM (Zanoveli et al., 2010; Roncon et al., 2012). Nevertheless, the specific opioid receptor involved in the anti-escape effect of fluoxetine is still unknown. Therefore, the aim of the present study was to verify the involvement of MORs in the effects of fluoxetine in rats subjected to the ETM. For this, we determined whether the blockade of MORs with CTOP, which was injected in the dPAG, would antagonize the anti-escape effect of fluoxetine, which was administered intraperitoneally (i.p.), daily for 21 days. Once this result was obtained, the next step was to investigate whether activation of MORs in the dPAG would hasten and/or facilitate the anti-escape effect of fluoxetine in the ETM.
2. Methods and materials 2.1. Animals Male Wistar rats (230e300 g) from the State University of and from the Ribeira ~o Preto Medical School of the UniMaringa versity of S~ ao Paulo (FMRP-USP) were housed in groups of five per cage in Plexiglas-walled cages inside a room maintained at 22 ± 1 C, with an alternating 12 h/12 h light/dark cycle (lights on from 07:00e19:00 h), and with free access to food and water except during testing. The experimental procedures adopted were approved by the State University of Maring a Committee of Ethical Conduct in the Use of Animals in Experiments (172/2013) and by ~o Preto the Experimental Animal Ethics Committee of Ribeira Medical School of the University of S~ ao Paulo (153/2012). Efforts were made to minimize animal suffering and to reduce the number of animals employed.
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2.2. Apparatus The ETM was made of wood and had three arms of equal dimensions (50 12 cm). One of the arms was enclosed by 40 cmhigh walls and was perpendicular to two opposite open arms. To avoid falls, the open arms were surrounded by a 1 cm high Plexiglas rim. The open field used to measure the putative effect of drug treatments on locomotion was made of wood and had a diameter 70 cm and was surrounded by 30 cm-high walls. Both apparatuses were elevated 50 cm above the floor. Brightness at the level of the maze arms and the open field was 60 lux. 2.3. Drugs The following drugs were used: fluoxetine hydrochloride (EMS, Brazil), D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP, Sigma Aldrich, USA) and [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt (DAMGO, Sigma Aldrich, USA). Fluoxetine was dissolved in a solution containing physiological saline (0.9% NaCl) with 2% Tween 80. CTOP and DAMGO were dissolved in sterile saline. All drugs were freshly prepared 30 min before the injections. 2.4. Surgery Rats were anesthetized with an intramuscular (i.m.) injection of ~o Química, Brazil) and xylazine (12 mg/ ketamine (60 mg/kg; Unia kg; Bayer, Brazil), and fixed to a stereotaxic frame (David Kopf, USA) for the implantation of a guide cannula in the dPAG. Before the implantation of the stainless steel guide cannula, the rats received local anesthesia with 2% lidocaine (Hipolabor, Brazil). The cannula (12 mm long; outer and inner diameter of 0.6 and 0.4 mm, respectively) was implanted following the coordinates from the atlas of Paxinos and Watson (2006): antero-posterior ¼ 6.6 mm from bregma, medio-lateral ¼ 2.0 mm, and dorsoventral ¼ 3.6 mm, at an angle of 24 with the sagittal plane. The guide cannula was fixed to the skull with acrylic resin and one stainless steel screw. The cannula was sealed with a stainless steel wire to avoid obstruction. After surgery, the rats were treated with 0.1 mL/100 g (i.m.) of an antibiotic (streptomycin and benzylpenicillin; Fort Dodge, USA) to prevent infections, and with 0.05 mL/ 100 g (s.c.) of the antiinflammatory flunixin meglumine (Banamine; Mantecorp, Brazil). 2.5. Microinjection of drugs Both DAMGO and CTOP were injected in the dPAG. A needle (0.3 mm outer diameter, 13.6 mm long) was introduced through the guide cannula until its tip was 1.6 mm below the end of the cannula. A volume of 0.2 mL was injected over a period of 120 s using a 10 mL microsyringe (Hamilton 701-RN) that was attached to a microinfusion pump (Stoelting, USA). The displacement of an air bubble inside the polyethylene catheter that connected the syringe needle to the intracerebral needle was used to monitor the microinjection. The intracerebral needle was removed 60 s after the end of the injection. 2.6. Procedure Six days after surgery, the rats were pre-exposed to one of the open arms of the ETM for 30 min, as described by Teixeira et al. (2000). A wooden barrier mounted between the central area of the maze and the proximal end of the arm closed the open arm's entry. Such pre-exposure has been shown to make the escape task more sensitive to anti-panic drugs because it shortens the latencies of escape from the open arm during the test. Twenty-four hours
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later, the ETM and the open-field tests were performed. For subchronic treatment, the rats were treated with fluoxetine or vehicle daily for 7 days, pre-exposed to the open arm of the ETM on the 6th day, and subjected to the ETM and open-field tests on the 7th day. For chronic treatment, the rats were treated with fluoxetine or vehicle daily for 21 days, pre-exposed to the open arm of the ETM test on the 20th day, and subjected to the ETM and open-field tests on the 21st day. In experiment 1, we investigated whether the activation of MORs in the dPAG mediates the effects of fluoxetine in the ETM. To this end, the rats were treated with i.p. injection of fluoxetine (10 mg/kg, 1 mL/kg) or vehicle solution daily for 21 days. After randomization, 10 min before the last injection of fluoxetine or vehicle solution, the rats were pre-treated with intra-dPAG microinjection of CTOP (1 nmol) or sterile saline, forming the following groups: saline intra-dPAG/vehicle i.p. (n ¼ 7), CTOP intradPAG/vehicle i.p. (n ¼ 6), saline intra-dPAG/fluoxetine i.p. (n ¼ 7) and CTOP intra-dPAG/fluoxetine i.p. (n ¼ 8). Thirty minutes after the last injection of fluoxetine or vehicle, the rats were subjected to the behavioral tests. The doses of fluoxetine and CTOP were chosen based on previous studies performed by our team using the same behavioral test (Zanoveli et al., 2007; Roncon et al., 2012). In experiment 2, doseeeffect curves were obtained to address the effects caused by a short-term administration fluoxetine and of DAMGO microinjected in the dPAG. Regarding fluoxetine, the rats were injected daily for 7 days, with either 5 or 10 mg/kg fluoxetine or with vehicle solution. On the test day, the rats received fluoxetine or vehicle solution (n ¼ 8e11) 30 min before being tested in the ETM and open field. Regarding DAMGO, the rats were intra-dPAG treated with only one injection of DAMGO at different doses (0.1, 0.3 or 0.5 nmol) or with saline (n ¼ 4e5); after 10 min they were subjected to the behavioral tests. To assess whether the activation of MORs in the dPAG accelerates the anti-escape effect of fluoxetine in the ETM, in experiment 3A, the rats were i.p. injected with 5 mg/kg fluoxetine or with vehicle solution daily for 7 days. After randomization, the rats received an intra-dPAG microinjection of a sub-effective dose of DAMGO (0.1 nmol) or of saline 20 min after the last injection of fluoxetine or vehicle solution, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 6), vehicle i.p./DAMGO intradPAG (n ¼ 6), fluoxetine 5 mg/kg i.p./saline intra-dPAG (n ¼ 7) and fluoxetine 5 mg/kg i.p./DAMGO intra-dPAG (n ¼ 6). In experiment 3B, the same experimental protocol was adopted, with the exception that instead of 5 mg/kg fluoxetine, the rats were treated with 10 mg/kg fluoxetine, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 5), vehicle i.p./DAMGO intra-dPAG (n ¼ 5), fluoxetine 10 mg/kg i.p./saline intra-dPAG (n ¼ 6) and fluoxetine 10 mg/kg i.p./DAMGO intra-dPAG (n ¼ 5). In experiments 3A and 3B, the rats were subjected to the behavioral tests 30 min after the last injection of fluoxetine or vehicle. In experiment 4, we investigated whether the activation of MORs in the dPAG enhances the response of a sub-effective dose of chronic fluoxetine treatment. To this end, the rats were i.p. injected with 5 mg/kg fluoxetine or with vehicle solution daily for 21 days. After randomization, the rats were intra-dPAG microinjected with DAMGO (0.1 nmol) or with saline 20 min after the last injection of fluoxetine or vehicle solution, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 6), vehicle i.p./DAMGO intradPAG (n ¼ 5), fluoxetine i.p./salina intra-dPAG (n ¼ 6) and fluoxetine i.p./DAMGO intra-dPAG (n ¼ 7). Thirty minutes after the last injection of fluoxetine or vehicle, the rats were subjected to the behavioral tests. The ETM is an apparatus made of an arm enclosed by walls perpendicular to two open arms, all elevated from the ground. Two behavioral responses are displayed in sequence by the same
experimental rat: the first one consists in avoiding the exploration of the open arms (avoidance response), and the second consists in an escape response from the open arms (escape reaction). For the measurement of the avoidance response, each rat was placed at the distal end of the enclosed arm of the ETM facing the intersection. The time taken by each rat to leave the enclosed arm with four paws was recorded in three consecutive trials (baseline, avoidance 1 and avoidance 2), with 30 s inter-trial intervals. When first placed at the end of the enclosed arm, the rat does not see the open arms until it pokes its head beyond the walls of the closed arm. Being on the open arm seems to be an aversive experience, since rats have an innate fear of open space (Treit et al., 1993). As such, in the two succeeding trials the animal gradually leaves the enclosed arm with longer latencies, indicating the acquisition of avoidance to this aversive stimulus. Following the avoidance task (30 s after), each rat was placed at the end of the same open arm used in the pre-exposure session, and the latency to leave this arm with four paws was recorded in three consecutive trials (escape 1, 2, and 3), again with 30 s inter-trial intervals. Contrary to what happens in the enclosed arm, the latency to leave the open arm usually does not change with successive trials (Zangrossi Jr. and Graeff, 2014). A cut-off time of 300 s was established for the avoidance and escape latencies. Immediately after the ETM test, the rat was placed inside the open field and allowed to freely explore the environment for 5 min to evaluate locomotor activity. The total distance travelled was analyzed using a video tracking system (Ethovision; Noldus, The Netherlands). 2.7. Histology After the pharmacological experiments, the rats were anesthetized with urethane 25% (0.5 mL/100 g; 45 mg/kg, i.p.) and perfused through the left ventricle of the heart with saline (0.9% NaCl) followed by 10% formalin solution. After that, 0.2 mL of methylene blue (2%) was microinjected into the dPAG to mark the site of drug injection. Brains were removed from the skull and maintained in 10% formalin. Serial coronal brain sections (60 mm) were cut with a cryostat (HM 505 Microm, Zeiss, Germany), mounted on gelatin-coated slides, and stained with neutral red. Only rats with injection sites located inside the dPAG, which includes the dorsolateral and dorsomedial columns of the PAG, were included in the statistical analysis. Misplacement of the guide cannula was found in 25% of the animals tested. 2.8. Statistical analysis Repeated-measures analysis of variance (R-ANOVA) was used to analyze both avoidance and escape data from the ETM. The systemic and central treatments were the independent factors, and trials (baseline, avoidance 1 and 2, and escape 1e3) were the repeated measures. However, because no effect of trials was detected in the escape task, latencies were merged, and the data from each rat were analyzed as the mean ± standard error of the mean (S.E.M.) of the three trials performed. Two-way analysis of variance was used to analyze this measure and the locomotion data. When appropriate, the Duncan post-hoc test was used. Significance level was set at p < 0.05. 3. Results The histologically confirmed sites of dPAG drug microinjection are shown in Fig. 1.
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Fig. 1. Diagrammatic representation of coronal sections of the rat midbrain showing the drug injection sites (dark circles) within the dorsal periaqueductal gray (dPAG). The figures represent coordinates from the Paxinos and Watson (2006) rat brain atlas. The number of points shown is fewer than the total number of rats used because of several instances of overlap. In experiment 1, 10 out of 38 rats tested were excluded for cannula misplacement. In experiment 2, 15 out of 62; experiment 3A, 7 out of 28; experiment 3B, 9 out of 34 and experiment 4, 6 out of 30. dmPAG, dorsomedial column of the periaqueductal gray matter; dlPAG, dorsolateral column of the periaqueductal gray matter.
3.1. Experiment 1: antagonism of the anti-escape effect of chronic fluoxetine by intra-dPAG CTOP
3.3. Experiment 3A: combined administration of intra-dPAG DAMGO and subchronic fluoxetine (5 mg/kg)
Chronic treatment with fluoxetine impaired escape performance in the ETM test, and this panicolytic-like effect was blocked by intra-dPAG injection of CTOP, as shown in Fig. 2. Two-way ANOVA revealed significant effects of systemic (F(1,80) ¼ 14.64; p < 0.05) and central (F(1,80) ¼ 11.29; p < 0.05) treatments, and an interaction between these two factors (F(1,80) ¼ 15.70; p < 0.05). None of the treatments employed affected avoidance acquisition in the ETM or locomotion in the open field (Table 1).
Fig. 3A shows that DAMGO (0.1 nmol) administration in the dPAG of animals treated with fluoxetine (5 mg/kg) for 7 days did not interfere with escape performance in the ETM. Neither avoidance response nor locomotion in the open field was changed by the treatments employed in this experiment (Table 1).
3.2. Experiment 2: effects of subchronic fluoxetine and intra-dPAG DAMGO Table 2 shows that whereas systemic injection of fluoxetine for 7 days did not affect escape expression, intra-dPAG injection of DAMGO inhibited this behavior (F(3,53) ¼ 9.50; p < 0.05). Neither avoidance response in the ETM nor the total distance travelled in the open field was affected by any of the drug treatments used (Table 2).
3.4. Experiment 3B: combined administration of intra-dPAG DAMGO and subchronic fluoxetine (10 mg/kg) Intra-dPAG administration of DAMGO (0.1 nmol) in rats subchronically treated with fluoxetine (10 mg/kg) impaired escape performance and facilitated avoidance acquisition in the ETM, as shown in Fig. 3B and Table 1, respectively. Regarding avoidance response, statistical analysis revealed significant main effects of trial (F(2,34) ¼ 8.76; p < 0.05) and of intra-dPAG treatment (F(1,17) ¼ 5.64; p < 0.05). Regarding escape, two-way ANOVA revealed significant effects of systemic (F(1,59) ¼ 19.74; p < 0.05) and central (F(1,59) ¼ 4.55; p < 0.05) treatments, and an interaction between these two factors (F(1,59) ¼ 6.19; p < 0.05). None of the treatments employed affected locomotion in the open field (Table 1). 3.5. Experiment 4: combined administration of intra-dPAG DAMGO and chronic fluoxetine (5 mg/kg) The administration of DAMGO in the dPAG (0.1 nmol) of rats treated with fluoxetine (5 mg/kg) for 21 days impaired escape performance and facilitated avoidance acquisition, as shown in Fig. 4 and Table 1, respectively. Concerning the avoidance response, R-ANOVA showed significant effects of trial (F(2,40) ¼ 24.32; p < 0.05) and of intra-dPAG treatment with DAMGO (central treatment e F(1,20) ¼ 7.49; p < 0.05). Regarding escape, two-way ANOVA showed significant effects of systemic (F(1,68) ¼ 17.75; p < 0.05) and central (F(1,68) ¼ 10.28; p < 0.05) treatments, and an interaction between these two factors (F(1,68) ¼ 8.67; p < 0.05). Locomotion in the open field did not differ among the groups tested here (Table 1).
Fig. 2. Pre-treatment with CTOP (1 nmol/0.2 mL) blocked the anti-escape effect of chronic fluoxetine administration (10 mg/kg, i.p., daily, 21 days) on escape performance in the ETM. Columns represent the mean; bars, the standard error of the mean (SEM). The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 6e8, *p < 0.05 compared to the saline þ vehicle group; #p < 0.05 compared to the saline þ fluoxetine group.
4. Discussion The present results showed that microinjection of the selective MOR antagonist CTOP in the dPAG blocked the anti-escape effect
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Table 1 Latency (mean ± standard error of the mean) in seconds to withdraw from the enclosed arm (avoidance) of the elevated T-maze (ETM) and total distance travelled in the open field by rats. *p < 0.05 compared to the vehicle þ physiological saline-treated groups, according to repeated measure ANOVA followed by Duncan's post hoc test. Drug treatment
N
Experiment 1 Saline þ Vehicle CTOP þ Vehicle Saline þ Fluo 10 CTOP þ Fluo 10 Experiment 3A Vehicle þ Saline Vehicle þ DAMGO Fluo 5 þ Saline Fluo 5 þ DAMGO Experiment 3B Vehicle þ Saline Vehicle þ DAMGO Fluo 10 þ Saline Fluo 10 þ DAMGO Experiment 4 Vehicle þ Saline Vehicle þ DAMGO Fluo 5 þ Saline Fluo 5 þ DAMGO
Baseline (s)
Avoidance 1 (s)
Avoidance 2 (s)
Total distance travelled (m)
7 6 7 8
11.85 77.16 57.42 59.62
± ± ± ±
2.83 47.65 40.53 36.09
81.00 113.00 125.57 55.12
± ± ± ±
43.46 59.45 47.70 35.16
156.42 131.83 175.00 76.75
± ± ± ±
54.18 53.93 45.92 34.85
20.35 16.11 19.30 17.93
± ± ± ±
1.55 2.59 2.61 1.87
6 6 7 6
22.55 18.00 27.57 17.83
± ± ± ±
6.89 5.70 12.16 8.26
76.33 30.00 86.85 68.33
± ± ± ±
25.98 13.68 38.96 46.78
123.50 128.66 154.00 117.50
± ± ± ±
38.98 56.88 50.22 58.90
19.72 16.51 20.76 18.29
± ± ± ±
1.44 1.32 1.28 1.64
5 5 6 5
10.20 24.20 12.50 47.20
± ± ± ±
2.35 4.85 5.07 26.00
25.20 119.00 64.50 176.20
± ± ± ±
9.17 53.09 47.20 58.63*
84.00 92.60 66.00 195.40
± ± ± ±
26.32 56.17 16.13 64.09
17.60 12.49 19.94 18.69
± ± ± ±
1.58 1.49 2.17 5.27
6 5 6 7
67.50 140.00 64.16 73.57
± ± ± ±
47.11 66.00 47.21 39.82
131.33 202.00 73.16 277.85
± ± ± ±
50.98 60.15 45.98 22.14*
176.33 300.00 160.66 289.42
± ± ± ±
56.28 41.80 50.02 10.57
16.39 11.58 16.83 14.04
± ± ± ±
2.44 0.80 2.02 1.80
caused by chronic systemic administration of fluoxetine. Previously reported evidence has implicated opioid receptors in the panicolytic-like effect of the same selective serotonin reuptake inhibitor (SSRI). More precisely, the anti-panic effect of chronic fluoxetine treatment in rodents subjected to the ETM was shown to be antagonized by intra-dPAG microinjection the nonselective opioid receptor antagonist naloxone (Roncon et al., 2012). Adding to the relevance of MORs in the anti-escape effect of fluoxetine, the results of experiment 3B showed that intra-dPAG
pre-treatment with a sub-effective dose of DAMGO (0.1 nmol) accelerated the anti-escape effect of this SSRI. Thus, in combination with this selective MOR agonist 10 mg/kg fluoxetine impaired escape expression after only 7 days of continuous systemic administration. This effect was not observed with the lower dose (5 mg/kg) of the SSRI (experiment 3A). Moreover, as shown in experiment 4, although the prolonged administration of fluoxetine (5 mg/kg) for 21 days failed to change the escape expression, the combination of this treatment with a single injection of 0.1 nmol of
Table 2 Latency (mean ± standard error of the mean) in seconds to withdraw from the enclosed arm (avoidance) and from the open arm (escape) of the elevated T-maze (ETM) and total distance travelled in the circular arena (open-field test) by rats tested in experiment 2. *p < 0.05 compared to the physiological saline-treated group *p < 0.05 compared to the saline group, according to repeated measure ANOVA followed by Duncan's post hoc test. Drug treatment
N
Baseline (s)
Vehicle Fluoxetine 5 Fluoxetine 10 Saline DAMGO 0.1 DAMGO 0.3 DAMGO 0.5
11 8 8 5 5 5 4
16.54 13.87 84.37 31.80 32.80 11.60 35.75
± ± ± ± ± ± ±
3.62 1.98 47.11 20.39 17.66 1.88 27.77
Avoidance 1 (s) 57.54 44.12 143.62 81.40 101.60 56.80 40.25
± ± ± ± ± ± ±
26.44 15.56 40.31 54.88 58.00 34.44 27.01
Avoidance 2 (s) 102.18 150.87 264.00 115.00 148.20 92.80 104.25
± ± ± ± ± ± ±
30.47 49.37 23.76 48.56 65.47 38.15 66.91
Escape (s) 9.54 11.33 15.16 6.80 7.86 13.80 16.25
± ± ± ± ± ± ±
0.73 1.10 2.31 0.61 1.51 1.75* 1.68*
Total distance travelled (m) 17.34 15.71 15.53 14.63 14.99 14.93 13.49
± ± ± ± ± ± ±
1.75 1.51 1.39 0.66 2.67 2.16 2.38
Fig. 3. (A) Combined administration of DAMGO (0.1 nmol) and subchronic administration of fluoxetine (5 mg/kg, i.p., daily, 7 days); (B) Anti-escape effect caused by the combined administration of DAMGO (0.1 nmol) and subchronic administration of fluoxetine (10 mg/kg, i.p., daily, 7 days). Columns represent the mean; bars, the SEM. The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 5e7. *p < 0.05 compared to the saline þ vehicle group; #P < 0.05 compared to the saline þ fluoxetine group.
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Fig. 4. Anti-escape effect caused by the combined administration of DAMGO (0.1 nmol) and chronic administration of fluoxetine (Fluo: 5 mg/kg, i.p., daily, 21 days). Columns represent the mean; bars, the SEM. The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 5e7. *p < 0.05 compared to the saline þ vehicle group; #p < 0.05 compared to the saline þ fluoxetine group.
DAMGO in the dPAG, a dose that was shown to be sub-effective in experiment 3, caused a clear panicolytic-like effect. This result provides further indication that chronic treatment with fluoxetine facilitates MOR-mediated neurotransmission in the dPAG. Consistent with these findings, chronic i.p. treatment (14 days) with fluoxetine (6 mg/kg) or imipramine (15 mg/kg) has been shown to increase the density of neurons expressing MOR in forebrain regions, such as the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices, suggesting that repeated administration of these antidepressants improves MOR neurotransmission in these areas (De Gandarias et al., 1998, 1999). It is noteworthy, however, that this effect of antidepressants may be related to the type of drug used and to the region investigated. For instance, an radioautographic study failed to show this facilitatory effect on cortical MORs of rats chronically treated (21 days) with other antidepressants, such as the SSRI paroxetine, the noradrenaline reuptake blocker reboxetine and the MAO inhibitor moclobemide. While paroxetine was shown to increase the density of MORs in the olfactory tubercle in the same study, it also decreased MOR density in thalamic sub-areas (Vilpoux et al., 2002). Recently gathered evidence by our research group has shown that prior administration of the 5-HT1A-R antagonist WAY-100635 in the dPAG blocks the anti-escape effect caused by 21 days of systemic fluoxetine treatment (Zanoveli et al., 2010), as observed here with the intra-dPAG microinjection of CTOP. Most importantly, reported evidence indicates that in the dPAG, MORs and 5-HT1A-Rs act cooperatively in the regulation of escape expression. Thus, while the inhibitory effect observed in the ETM escape task after intra-dPAG injection of the 5-HT1A-R agonist 8-OH-DPAT is blocked by previous local administration of CTOP, blockade of 5-HT1A-R by WAY-100635 reciprocally antagonizes the anti-escape effect of DAMGO when both drugs are microinjected in the dPAG (Roncon et al., 2013). In agreement with our findings, Kishimoto et al. (2001) showed that MORs and the 5-HT1A-Rs act synergistically in rat midbrain neurons, more specifically in the ventrolateral PAG. According to their results, activation of both MORs and 5-HT1A-Rs on GABAergic presynaptic neural terminals inhibited GABA release. The signaling pathways responsible for the presynaptic modulation elicited by these two receptor types share a common intracellular mechanism, the opening of Kþ channels via PTX-sensitive G proteins. Subthreshold DAMGO concentrations increased presynaptic serotonergic inhibition and had a significant inhibitory effect when combined with subthreshold concentrations of serotonin. Taken
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together, these results suggest presynaptic synergism between MORs and 5-HT1A-Rs in the ventrolateral PAG. A wealth of evidence obtained over the last several years indicates that chronic (21 days), but not acute or short-term (3 days), fluoxetine administration (10 mg/kg) not only increases 5-HT release in the dPAG, as measured by in vivo microdialysis (Zanoveli et al., 2010), but also enhances the functional reactivity of 5-HT1A-R in this midbrain area, suggesting that sensitization of these receptors may have occurred (De Bortoli et al., 2006; Zanoveli et al., 2007). As mentioned above, the results of experiment 3B further indicate that chronic fluoxetine administration increases the responsiveness of MORs in the dPAG. Given the evidence that these receptors may work cooperatively, forming heterodimers, whether the facilitatory changes induced by this SSRI in the functioning of these receptors are temporally related and how long they last after drug discontinuation remain to be explored. One intriguing finding of the current study was that avoidance of the open arms was facilitated by DAMGO injection in the dPAG, but only in animals that were treated with fluoxetine for either 7 (10 mg/kg) or 21 (5 mg/kg) days. It is important to say that, in these animals, the combined treatment of fluoxetine and DAMGO also caused an anti-escape effect, as discussed above. As both avoidance and escape latencies were increased in these experiments, a motor deficit may be one possible explanation for the obtained results. However, the different treatments presently used here did not significantly modify the total distance traveled by the animals in the open-field test. This result indicates that the effects of these drugs in the ETM were not due to non-specific alterations in motor function. A possible explanation is that the synergic interaction between 5-HT1A-Rs and MORs may have also influenced the functioning of other 5-HT or opioid receptors in the same brain area. In a recently published paper, De Melo Yamashita et al. (2011) observed that intra-dPAG injection of the 5-HT2C receptor agonist MK-212 facilitated avoidance acquisition in rodents subjected to the ETM, indicating an anxiogenic-like effect, without interfering with escape expression. This anxiogenic-like effect was blocked by local microinjection of the 5-HT2C receptor antagonist SB-242084. Therefore, facilitation of 5-HT2C receptor activity in the dPAG seems to interfere with behaviors associated with anxiety, but not with panic. It is also worthy of note that the activation of kappa receptors in the same midbrain area has anxiogenic consequences (Motta et al., 1995), but there is no evidence to date indicating that such activation would not impact the expression of panic-related defensive behaviors. 5. Conclusion The present work provides pharmacological evidence for the involvement of MORs in the mediation of fluoxetine effects. They also indicate that the use of opioids in combination with antidepressants may be a relevant strategy to augment and hasten the onset of the therapeutic effects of antidepressants. Acknowledgments Pesquisa do This work was supported by Fundaç~ ao de Amparo a ~o Paulo (FAPESP) (grant 2012/03798-0), Conselho Estado de Sa gico (CNPq) Nacional de Desenvolvimento Científico e Tecnolo (grant 470119/2004-7), Fundaç~ ao de Apoio ao Ensino, Pesquisa e ^ncia do HC-FMRP-USP (FAEPA) (grant 1291/97, 355/2000, Assiste ~o 68/2001 and 15/2003), and a Pro-Rectory of the University of Sa Paulo (USP) Research grant (IaPQ2012; NAP-USP-NuPNE-156). Each organization had no further role in the design of the study; in the collection, analysis and interpretation of the data; in the writing of
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the report; or in the decision to submit the paper for publication. C.M. Roncon is the recipient of a post-doctorate fellowship from FAPESP (process 2012/23238-0) and was involved in the design of the study, performed the behavioral and, analyzed the results and wrote the manuscript. R.C. Almada participated in the data analysis. J. G. Maraschin collaborated in the psychopharmacological experiments. N.C. Coimbra, F. G. Graeff, H. Zangrossi Jr., and E. A. Audi conceived the study, contributed to the analysis of data, and drafted and revised the final manuscript. All authors read and approved the final version of the manuscript. References Almada, R.C., Coimbra, N.C., 2015. Recruitment of striatonigral disinhibitory and nigrotectal inhibitory GABAergic pathways during the organization of defensive behaviour by mice in a dangerous environment with the venomous snake Bothrops alternatus (Reptilia, Viperidae). Synapse 69, 299e313. Almada, R.C., Roncon, C.M., Elias-Filho, D.H., Coimbra, N.C., 2015. Endocannabinoid signaling mechanisms in the substantia nigra pars reticulata modulate GABAergic nigro-tectal pathways in mice threatened by urutu-cruzeiro venomous pit viper. Neuroscience 303, 503e514. Amano, K., Tanikawa, T., Iseki, H., Kawabatake, H., Notani, M., Kawamura, H., Kitamura, K., 1978. Single neuron analysis of the human midbrain tegmentum. Rostral mesencephalic reticulotomy for pain relief. Appl. Neuropsychol. 41, 66e78. Bandler, R., Shipley, M.T., 1994. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? TINS 17, 379e389. Coimbra, N.C., De Oliveira, R., Freitas, R.L., Ribeiro, S.J., Borelli, K.G., Pacagnella, R.C., ~o, M.L., 2006. Moreira, J.E., da Silva, L.A., Melo, L.L., Lunardi, L.O., Branda Neuroanatomical approaches of the tectum-reticular pathways and immunohistochemical evidence for serotonin-positive perikarya on neuronal substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear-induced analgesia. Exp. Neurol. 197, 93e112. Deakin, J.F.W., Graeff, F.G., 1991. 5-HT and mechanisms of defence. J. Psychopharmacol. 5, 305e315. De Bortoli, V.C., Nogueira, R.L., Zangrossi Jr., H., 2006. Effects of fluoxetine and buspirone on the panicolytic-like response induced by the activation of 5-HT1A and 5-HT2A receptors in the rat dorsal periaqueductal gray. Psychopharmacology 183, 422e428. De Gandarias, J.M., Echevarria, E., Acebes, I., Silio, M., Casis, L., 1998. Effects of imipramine administration on m-opioid receptor immunostaining in the rat forebrain. Arzneimittelforschung 48, 717e719. De Gandarias, J.M., Echevarría, E., Acebes, I., Abecia, L.C., Casis, O., Casis, L., 1999. Effects of fluoxetine administration on m-opioid receptor immunostaining in the rat forebrain. Brain Res. 817, 236e240. Del-Ben, C.M., Graeff, F.G., 2009. Panic disorder: is the PAG involved? Neural Plast. 2009, 1e9. De Melo Yamashita, P.S., De Bortoli, V.C., Zangrossi Jr., H., 2011. 5-HT2C receptor regulation of defensive responses in the rat dorsal periaqueductal gray. Neuropharmacology 60, 216e222. Eichenberger, G.C.D., Ribeiro, S.J., Osaki, M.Y., Maruoka, R.Y., Resende, G.C.C., ^a, S.A.L., Da Silva, L.A., Coimbra, N.C., 2002. NeuroCastellan-Baldan, L., Corre anatomical and psychopharmacological evidence for interaction between opioid and GABAergic neuronal pathways in the modulation of fear and defense elicited by electrical and chemical stimulation of deep layers of the superior colliculus and dorsal periaqueductal gray matter. Neuropharmacology 42, 48e59. Fanselow, M.S., 1991. The Midbrain Periaqueductal Gray as a Coordinator of Action in Response to Fear and Anxiety. NATO ASI Series, vol. 213, pp. 151e173. Fanselow, M.S., Lester, L.S., 1988. A functional behavioristic approach to aversively motivated behavior: Predatory imminence as a determinant of the topography of defensive behavior. In: Bolles, R.C., Beecher, M.D. (Eds.), Evolution and Learning. Erlbaum, Hillsdale, N. J, pp. 185e211. Graeff, F.G., 1990. Brain defense systems and anxiety. In: Burrows, G.D., Roth, M., Noyers Jr., (Eds.), Handbook of anxiety. Elsevier Science Publishers, Amsterdam, pp. 307e354. Graeff, F.G., 1994. Neuroanatomy and neurotransmitter regulation of defensive behaviors and related emotions in mammals. Braz. J. Med. Biol. Res. 27, 811e829. Graeff, F.G., Netto Ferreira, C., Zangrossi Jr., H., 1998. The elevated T-maze as an experimental model of anxiety. Neurosci. Biobehav. Rev. 23, 237e246. Graeff, F.G., 2004. Serotonin, the periaqueductal gray and panic disorder. Neurosci. Biobehav. Rev. 28, 239e259. Graeff, F.G., Zangrossi Jr., H., 2010. The dual role of serotonin in defense and the mode of action of antidepressants on generalized anxiety and panic disorders.
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Pharmacological evidence for the mediation of the panicolytic effect of fluoxetine by dorsal periaqueductal gray matter m-opioid receptors Camila Marroni Roncon a, d, Rafael Carvalho Almada a, d, Jhonatan Christian Maraschin b, lio Zangrossi Jr. c, e, Frederico Guilherme Graeff d, e, Elisabeth Aparecida Audi b, He a, d, e, * Norberto Cysne Coimbra ~o Preto Medical School of the University of Sa ~o Paulo, Av. dos Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeira ~o Preto, Sa ~o Paulo 14049-900, Brazil Bandeirantes, 3900, Ribeira , Av. Colombo, 5790, Maringa , Parana Laboratory of Psychopharmacology, Department of Pharmacology and Therapeutics, State University of Maringa 87020-900, Brazil c ~o Preto Medical School of the University of Sa ~o Paulo, Av. dos Bandeirantes, 3900, Ribeira ~o Preto, Sa ~o Paulo 14049Department of Pharmacology, Ribeira 900, Brazil d ~o Preto, Sa ~o Paulo 14050-220, Brazil Behavioural Neurosciences Institute (INeC), Av. do Caf e, 2450, Monte Alegre, Ribeira e ~o Preto School of Medicine of the University of Sa ~o Paulo (FMRP-USP), Av. dos Neurobiology of Emotions Research Centre (NAP-USP-NuPNE), Ribeira ~o Preto, Sa ~o Paulo 14049-900, Brazil Bandeirantes, 3900, Ribeira a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2015 Received in revised form 20 August 2015 Accepted 21 August 2015 Available online 28 August 2015
Previously reported results have shown that the inhibitory effect of fluoxetine on escape behavior, interpreted as a panicolytic-like effect, is blocked by pretreatment with either the opioid receptor antagonist naloxone or the 5-HT1A receptor (5-HT1A-R) antagonist WAY100635 via injection into the dorsal periaqueductal gray matter (dPAG). Additionally, reported evidence indicates that the m-opioid receptor (MOR) interacts with the 5-HT1A-R in the dPAG. In the present work, pretreatment of the dPAG with the selective MOR blocker CTOP antagonized the anti-escape effect of chronic fluoxetine (10 mg/kg, i.p., daily, for 21 days), as measured in the elevated T-maze (ETM) test, indicating mediation of this effect by the MOR. In addition, the combined administration of sub-effective doses of the selective MOR agonist DAMGO (intra-dPAG) and sub-effective doses of chronic as well as subchronic (7 days) fluoxetine increased avoidance and escape latencies, suggesting that the activation of MORs may facilitate and accelerate the effects of fluoxetine. The current observation that MORs located in the dPAG mediate the anti-escape effect of fluoxetine may open new perspectives for the development of more efficient and fast-acting panic-alleviating drugs. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Dorsal periaqueductal gray matter m-opioid receptor 5-HT1A receptor Fluoxetine Elevated T-maze Panic
1. Introduction A wealth of reported evidence supports a key role of the dorsal periaqueductal gray matter (dPAG) in the regulation of both behavioral and neurovegetative manifestations of defensive reactions (Graeff, 1990, 1994; Bandler and Shipley, 1994; Lovick, 2000; Eichenberger et al., 2002; Ribeiro et al., 2005; Coimbra
Abbreviations: 5-HT1A-R, 5-HT1A receptor; dPAG, dorsal periaqueductal gray matter; MOR, m-opioid receptor; ETM, elevated T maze; 5-HT, serotonin. * Corresponding author. Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeir~ ao Preto Medical School of the University of ~o Preto, Sa ~o Paulo 14049-900, Brazil. S~ ao Paulo, Av. dos Bandeirantes, 3900, Ribeira E-mail address: [email protected] (N.C. Coimbra). http://dx.doi.org/10.1016/j.neuropharm.2015.08.037 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
et al., 2006; Twardowschy and Coimbra, 2015). Electrical or chemical stimulation of the dPAG in rodents causes behavioral changes characterized by vigorous, non-oriented escape reactions accompanied by autonomic responses, such as tachycardia, exophthalmia and increased blood pressure, which resemble those expressed in highly aversive situations such as during a predatory attack (Schutz et al., 1985; Jenck et al., 1995; Schenberg et al., 2001; Ullah et al., 2015; Almada and Coimbra, 2015; Almada et al., 2015). In humans, electrical stimulation of this structure evokes marked fear or terror, feelings of imminent death, and an urge to flee (Amano et al., 1978; Nashold et al., 1969). It has been suggested that the dPAG is fundamental for the organization of defensive behaviors related to coping with proximal threat/danger (Fanselow and Lester, 1988; Fanselow, 1991) and that malfunctioning of this
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mechanism may lead to increased susceptibility to the panic attacks that characterizes panic disorder (Deakin and Graeff, 1991; Del-Ben and Graeff, 2009; Graeff, 2004). There is substantial experimental evidence indicating that serotonin (5-HT) inhibits the escape response generated by stimulation of the dPAG (Graeff, 2004; Zanoveli et al., 2003, 2010; Graeff and Zangrossi Jr., 2010). In addition, recent studies suggest that 5HT and endogenous opioid peptides act together in the organization of defensive reactions evoked by proximal danger (Roncon et al., 2012, 2013; Rangel et al., 2014). Initially, it was shown that the selective 5-HT1A receptor (5-HT1A-R) antagonist WAY-100635 attenuated the anti-escape effect of low doses of the nonselective opioid receptor agonist morphine, suggesting a pharmacological interaction between 5-HT1A-Rs and endogenous opioids (Roncon et al., 2013). In this study, escape performance was measured in the elevated T-maze (ETM), which has been pharmacologically validated as an animal model of panic. The ETM also allows the measurement of avoidance, which has been related, in terms of psychopathology, to generalized anxiety disorder (Graeff et al, 1998; Pinheiro et al., 2007; Zangrossi Jr. et al, 2001; Moreira et al., 2013; Zangrossi Jr. and Graeff, 2014). Subsequently, it was demonstrated that the selective m-opioid receptor (MOR) antagonist CTOP attenuated the anti-escape effect of the selective 5-HT1AR agonist 8-OH-DPAT. In addition, sub-effective doses of both 8-OHDPAT and the selective MOR agonist DAMGO increased escape latencies in the elevated T-maze, which was interpreted as an antipanic effect. This set of information suggests that 5-HT1A-Rs and MORs in the dPAG interact cooperatively in the modulation of proximal defense (Roncon et al., 2013). The evidence cited above may be relevant for understanding the mechanism of action of anti-panic drugs. Thus, it has been shown that both WAY-100635 and the non-selective opioid receptor antagonist naloxone block the anti-escape effect of chronic treatment with the widely used anti-panic agent fluoxetine in the ETM (Zanoveli et al., 2010; Roncon et al., 2012). Nevertheless, the specific opioid receptor involved in the anti-escape effect of fluoxetine is still unknown. Therefore, the aim of the present study was to verify the involvement of MORs in the effects of fluoxetine in rats subjected to the ETM. For this, we determined whether the blockade of MORs with CTOP, which was injected in the dPAG, would antagonize the anti-escape effect of fluoxetine, which was administered intraperitoneally (i.p.), daily for 21 days. Once this result was obtained, the next step was to investigate whether activation of MORs in the dPAG would hasten and/or facilitate the anti-escape effect of fluoxetine in the ETM.
2. Methods and materials 2.1. Animals Male Wistar rats (230e300 g) from the State University of and from the Ribeira ~o Preto Medical School of the UniMaringa versity of S~ ao Paulo (FMRP-USP) were housed in groups of five per cage in Plexiglas-walled cages inside a room maintained at 22 ± 1 C, with an alternating 12 h/12 h light/dark cycle (lights on from 07:00e19:00 h), and with free access to food and water except during testing. The experimental procedures adopted were approved by the State University of Maring a Committee of Ethical Conduct in the Use of Animals in Experiments (172/2013) and by ~o Preto the Experimental Animal Ethics Committee of Ribeira Medical School of the University of S~ ao Paulo (153/2012). Efforts were made to minimize animal suffering and to reduce the number of animals employed.
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2.2. Apparatus The ETM was made of wood and had three arms of equal dimensions (50 12 cm). One of the arms was enclosed by 40 cmhigh walls and was perpendicular to two opposite open arms. To avoid falls, the open arms were surrounded by a 1 cm high Plexiglas rim. The open field used to measure the putative effect of drug treatments on locomotion was made of wood and had a diameter 70 cm and was surrounded by 30 cm-high walls. Both apparatuses were elevated 50 cm above the floor. Brightness at the level of the maze arms and the open field was 60 lux. 2.3. Drugs The following drugs were used: fluoxetine hydrochloride (EMS, Brazil), D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP, Sigma Aldrich, USA) and [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt (DAMGO, Sigma Aldrich, USA). Fluoxetine was dissolved in a solution containing physiological saline (0.9% NaCl) with 2% Tween 80. CTOP and DAMGO were dissolved in sterile saline. All drugs were freshly prepared 30 min before the injections. 2.4. Surgery Rats were anesthetized with an intramuscular (i.m.) injection of ~o Química, Brazil) and xylazine (12 mg/ ketamine (60 mg/kg; Unia kg; Bayer, Brazil), and fixed to a stereotaxic frame (David Kopf, USA) for the implantation of a guide cannula in the dPAG. Before the implantation of the stainless steel guide cannula, the rats received local anesthesia with 2% lidocaine (Hipolabor, Brazil). The cannula (12 mm long; outer and inner diameter of 0.6 and 0.4 mm, respectively) was implanted following the coordinates from the atlas of Paxinos and Watson (2006): antero-posterior ¼ 6.6 mm from bregma, medio-lateral ¼ 2.0 mm, and dorsoventral ¼ 3.6 mm, at an angle of 24 with the sagittal plane. The guide cannula was fixed to the skull with acrylic resin and one stainless steel screw. The cannula was sealed with a stainless steel wire to avoid obstruction. After surgery, the rats were treated with 0.1 mL/100 g (i.m.) of an antibiotic (streptomycin and benzylpenicillin; Fort Dodge, USA) to prevent infections, and with 0.05 mL/ 100 g (s.c.) of the antiinflammatory flunixin meglumine (Banamine; Mantecorp, Brazil). 2.5. Microinjection of drugs Both DAMGO and CTOP were injected in the dPAG. A needle (0.3 mm outer diameter, 13.6 mm long) was introduced through the guide cannula until its tip was 1.6 mm below the end of the cannula. A volume of 0.2 mL was injected over a period of 120 s using a 10 mL microsyringe (Hamilton 701-RN) that was attached to a microinfusion pump (Stoelting, USA). The displacement of an air bubble inside the polyethylene catheter that connected the syringe needle to the intracerebral needle was used to monitor the microinjection. The intracerebral needle was removed 60 s after the end of the injection. 2.6. Procedure Six days after surgery, the rats were pre-exposed to one of the open arms of the ETM for 30 min, as described by Teixeira et al. (2000). A wooden barrier mounted between the central area of the maze and the proximal end of the arm closed the open arm's entry. Such pre-exposure has been shown to make the escape task more sensitive to anti-panic drugs because it shortens the latencies of escape from the open arm during the test. Twenty-four hours
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later, the ETM and the open-field tests were performed. For subchronic treatment, the rats were treated with fluoxetine or vehicle daily for 7 days, pre-exposed to the open arm of the ETM on the 6th day, and subjected to the ETM and open-field tests on the 7th day. For chronic treatment, the rats were treated with fluoxetine or vehicle daily for 21 days, pre-exposed to the open arm of the ETM test on the 20th day, and subjected to the ETM and open-field tests on the 21st day. In experiment 1, we investigated whether the activation of MORs in the dPAG mediates the effects of fluoxetine in the ETM. To this end, the rats were treated with i.p. injection of fluoxetine (10 mg/kg, 1 mL/kg) or vehicle solution daily for 21 days. After randomization, 10 min before the last injection of fluoxetine or vehicle solution, the rats were pre-treated with intra-dPAG microinjection of CTOP (1 nmol) or sterile saline, forming the following groups: saline intra-dPAG/vehicle i.p. (n ¼ 7), CTOP intradPAG/vehicle i.p. (n ¼ 6), saline intra-dPAG/fluoxetine i.p. (n ¼ 7) and CTOP intra-dPAG/fluoxetine i.p. (n ¼ 8). Thirty minutes after the last injection of fluoxetine or vehicle, the rats were subjected to the behavioral tests. The doses of fluoxetine and CTOP were chosen based on previous studies performed by our team using the same behavioral test (Zanoveli et al., 2007; Roncon et al., 2012). In experiment 2, doseeeffect curves were obtained to address the effects caused by a short-term administration fluoxetine and of DAMGO microinjected in the dPAG. Regarding fluoxetine, the rats were injected daily for 7 days, with either 5 or 10 mg/kg fluoxetine or with vehicle solution. On the test day, the rats received fluoxetine or vehicle solution (n ¼ 8e11) 30 min before being tested in the ETM and open field. Regarding DAMGO, the rats were intra-dPAG treated with only one injection of DAMGO at different doses (0.1, 0.3 or 0.5 nmol) or with saline (n ¼ 4e5); after 10 min they were subjected to the behavioral tests. To assess whether the activation of MORs in the dPAG accelerates the anti-escape effect of fluoxetine in the ETM, in experiment 3A, the rats were i.p. injected with 5 mg/kg fluoxetine or with vehicle solution daily for 7 days. After randomization, the rats received an intra-dPAG microinjection of a sub-effective dose of DAMGO (0.1 nmol) or of saline 20 min after the last injection of fluoxetine or vehicle solution, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 6), vehicle i.p./DAMGO intradPAG (n ¼ 6), fluoxetine 5 mg/kg i.p./saline intra-dPAG (n ¼ 7) and fluoxetine 5 mg/kg i.p./DAMGO intra-dPAG (n ¼ 6). In experiment 3B, the same experimental protocol was adopted, with the exception that instead of 5 mg/kg fluoxetine, the rats were treated with 10 mg/kg fluoxetine, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 5), vehicle i.p./DAMGO intra-dPAG (n ¼ 5), fluoxetine 10 mg/kg i.p./saline intra-dPAG (n ¼ 6) and fluoxetine 10 mg/kg i.p./DAMGO intra-dPAG (n ¼ 5). In experiments 3A and 3B, the rats were subjected to the behavioral tests 30 min after the last injection of fluoxetine or vehicle. In experiment 4, we investigated whether the activation of MORs in the dPAG enhances the response of a sub-effective dose of chronic fluoxetine treatment. To this end, the rats were i.p. injected with 5 mg/kg fluoxetine or with vehicle solution daily for 21 days. After randomization, the rats were intra-dPAG microinjected with DAMGO (0.1 nmol) or with saline 20 min after the last injection of fluoxetine or vehicle solution, forming the following groups: vehicle i.p./saline intra-dPAG (n ¼ 6), vehicle i.p./DAMGO intradPAG (n ¼ 5), fluoxetine i.p./salina intra-dPAG (n ¼ 6) and fluoxetine i.p./DAMGO intra-dPAG (n ¼ 7). Thirty minutes after the last injection of fluoxetine or vehicle, the rats were subjected to the behavioral tests. The ETM is an apparatus made of an arm enclosed by walls perpendicular to two open arms, all elevated from the ground. Two behavioral responses are displayed in sequence by the same
experimental rat: the first one consists in avoiding the exploration of the open arms (avoidance response), and the second consists in an escape response from the open arms (escape reaction). For the measurement of the avoidance response, each rat was placed at the distal end of the enclosed arm of the ETM facing the intersection. The time taken by each rat to leave the enclosed arm with four paws was recorded in three consecutive trials (baseline, avoidance 1 and avoidance 2), with 30 s inter-trial intervals. When first placed at the end of the enclosed arm, the rat does not see the open arms until it pokes its head beyond the walls of the closed arm. Being on the open arm seems to be an aversive experience, since rats have an innate fear of open space (Treit et al., 1993). As such, in the two succeeding trials the animal gradually leaves the enclosed arm with longer latencies, indicating the acquisition of avoidance to this aversive stimulus. Following the avoidance task (30 s after), each rat was placed at the end of the same open arm used in the pre-exposure session, and the latency to leave this arm with four paws was recorded in three consecutive trials (escape 1, 2, and 3), again with 30 s inter-trial intervals. Contrary to what happens in the enclosed arm, the latency to leave the open arm usually does not change with successive trials (Zangrossi Jr. and Graeff, 2014). A cut-off time of 300 s was established for the avoidance and escape latencies. Immediately after the ETM test, the rat was placed inside the open field and allowed to freely explore the environment for 5 min to evaluate locomotor activity. The total distance travelled was analyzed using a video tracking system (Ethovision; Noldus, The Netherlands). 2.7. Histology After the pharmacological experiments, the rats were anesthetized with urethane 25% (0.5 mL/100 g; 45 mg/kg, i.p.) and perfused through the left ventricle of the heart with saline (0.9% NaCl) followed by 10% formalin solution. After that, 0.2 mL of methylene blue (2%) was microinjected into the dPAG to mark the site of drug injection. Brains were removed from the skull and maintained in 10% formalin. Serial coronal brain sections (60 mm) were cut with a cryostat (HM 505 Microm, Zeiss, Germany), mounted on gelatin-coated slides, and stained with neutral red. Only rats with injection sites located inside the dPAG, which includes the dorsolateral and dorsomedial columns of the PAG, were included in the statistical analysis. Misplacement of the guide cannula was found in 25% of the animals tested. 2.8. Statistical analysis Repeated-measures analysis of variance (R-ANOVA) was used to analyze both avoidance and escape data from the ETM. The systemic and central treatments were the independent factors, and trials (baseline, avoidance 1 and 2, and escape 1e3) were the repeated measures. However, because no effect of trials was detected in the escape task, latencies were merged, and the data from each rat were analyzed as the mean ± standard error of the mean (S.E.M.) of the three trials performed. Two-way analysis of variance was used to analyze this measure and the locomotion data. When appropriate, the Duncan post-hoc test was used. Significance level was set at p < 0.05. 3. Results The histologically confirmed sites of dPAG drug microinjection are shown in Fig. 1.
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Fig. 1. Diagrammatic representation of coronal sections of the rat midbrain showing the drug injection sites (dark circles) within the dorsal periaqueductal gray (dPAG). The figures represent coordinates from the Paxinos and Watson (2006) rat brain atlas. The number of points shown is fewer than the total number of rats used because of several instances of overlap. In experiment 1, 10 out of 38 rats tested were excluded for cannula misplacement. In experiment 2, 15 out of 62; experiment 3A, 7 out of 28; experiment 3B, 9 out of 34 and experiment 4, 6 out of 30. dmPAG, dorsomedial column of the periaqueductal gray matter; dlPAG, dorsolateral column of the periaqueductal gray matter.
3.1. Experiment 1: antagonism of the anti-escape effect of chronic fluoxetine by intra-dPAG CTOP
3.3. Experiment 3A: combined administration of intra-dPAG DAMGO and subchronic fluoxetine (5 mg/kg)
Chronic treatment with fluoxetine impaired escape performance in the ETM test, and this panicolytic-like effect was blocked by intra-dPAG injection of CTOP, as shown in Fig. 2. Two-way ANOVA revealed significant effects of systemic (F(1,80) ¼ 14.64; p < 0.05) and central (F(1,80) ¼ 11.29; p < 0.05) treatments, and an interaction between these two factors (F(1,80) ¼ 15.70; p < 0.05). None of the treatments employed affected avoidance acquisition in the ETM or locomotion in the open field (Table 1).
Fig. 3A shows that DAMGO (0.1 nmol) administration in the dPAG of animals treated with fluoxetine (5 mg/kg) for 7 days did not interfere with escape performance in the ETM. Neither avoidance response nor locomotion in the open field was changed by the treatments employed in this experiment (Table 1).
3.2. Experiment 2: effects of subchronic fluoxetine and intra-dPAG DAMGO Table 2 shows that whereas systemic injection of fluoxetine for 7 days did not affect escape expression, intra-dPAG injection of DAMGO inhibited this behavior (F(3,53) ¼ 9.50; p < 0.05). Neither avoidance response in the ETM nor the total distance travelled in the open field was affected by any of the drug treatments used (Table 2).
3.4. Experiment 3B: combined administration of intra-dPAG DAMGO and subchronic fluoxetine (10 mg/kg) Intra-dPAG administration of DAMGO (0.1 nmol) in rats subchronically treated with fluoxetine (10 mg/kg) impaired escape performance and facilitated avoidance acquisition in the ETM, as shown in Fig. 3B and Table 1, respectively. Regarding avoidance response, statistical analysis revealed significant main effects of trial (F(2,34) ¼ 8.76; p < 0.05) and of intra-dPAG treatment (F(1,17) ¼ 5.64; p < 0.05). Regarding escape, two-way ANOVA revealed significant effects of systemic (F(1,59) ¼ 19.74; p < 0.05) and central (F(1,59) ¼ 4.55; p < 0.05) treatments, and an interaction between these two factors (F(1,59) ¼ 6.19; p < 0.05). None of the treatments employed affected locomotion in the open field (Table 1). 3.5. Experiment 4: combined administration of intra-dPAG DAMGO and chronic fluoxetine (5 mg/kg) The administration of DAMGO in the dPAG (0.1 nmol) of rats treated with fluoxetine (5 mg/kg) for 21 days impaired escape performance and facilitated avoidance acquisition, as shown in Fig. 4 and Table 1, respectively. Concerning the avoidance response, R-ANOVA showed significant effects of trial (F(2,40) ¼ 24.32; p < 0.05) and of intra-dPAG treatment with DAMGO (central treatment e F(1,20) ¼ 7.49; p < 0.05). Regarding escape, two-way ANOVA showed significant effects of systemic (F(1,68) ¼ 17.75; p < 0.05) and central (F(1,68) ¼ 10.28; p < 0.05) treatments, and an interaction between these two factors (F(1,68) ¼ 8.67; p < 0.05). Locomotion in the open field did not differ among the groups tested here (Table 1).
Fig. 2. Pre-treatment with CTOP (1 nmol/0.2 mL) blocked the anti-escape effect of chronic fluoxetine administration (10 mg/kg, i.p., daily, 21 days) on escape performance in the ETM. Columns represent the mean; bars, the standard error of the mean (SEM). The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 6e8, *p < 0.05 compared to the saline þ vehicle group; #p < 0.05 compared to the saline þ fluoxetine group.
4. Discussion The present results showed that microinjection of the selective MOR antagonist CTOP in the dPAG blocked the anti-escape effect
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Table 1 Latency (mean ± standard error of the mean) in seconds to withdraw from the enclosed arm (avoidance) of the elevated T-maze (ETM) and total distance travelled in the open field by rats. *p < 0.05 compared to the vehicle þ physiological saline-treated groups, according to repeated measure ANOVA followed by Duncan's post hoc test. Drug treatment
N
Experiment 1 Saline þ Vehicle CTOP þ Vehicle Saline þ Fluo 10 CTOP þ Fluo 10 Experiment 3A Vehicle þ Saline Vehicle þ DAMGO Fluo 5 þ Saline Fluo 5 þ DAMGO Experiment 3B Vehicle þ Saline Vehicle þ DAMGO Fluo 10 þ Saline Fluo 10 þ DAMGO Experiment 4 Vehicle þ Saline Vehicle þ DAMGO Fluo 5 þ Saline Fluo 5 þ DAMGO
Baseline (s)
Avoidance 1 (s)
Avoidance 2 (s)
Total distance travelled (m)
7 6 7 8
11.85 77.16 57.42 59.62
± ± ± ±
2.83 47.65 40.53 36.09
81.00 113.00 125.57 55.12
± ± ± ±
43.46 59.45 47.70 35.16
156.42 131.83 175.00 76.75
± ± ± ±
54.18 53.93 45.92 34.85
20.35 16.11 19.30 17.93
± ± ± ±
1.55 2.59 2.61 1.87
6 6 7 6
22.55 18.00 27.57 17.83
± ± ± ±
6.89 5.70 12.16 8.26
76.33 30.00 86.85 68.33
± ± ± ±
25.98 13.68 38.96 46.78
123.50 128.66 154.00 117.50
± ± ± ±
38.98 56.88 50.22 58.90
19.72 16.51 20.76 18.29
± ± ± ±
1.44 1.32 1.28 1.64
5 5 6 5
10.20 24.20 12.50 47.20
± ± ± ±
2.35 4.85 5.07 26.00
25.20 119.00 64.50 176.20
± ± ± ±
9.17 53.09 47.20 58.63*
84.00 92.60 66.00 195.40
± ± ± ±
26.32 56.17 16.13 64.09
17.60 12.49 19.94 18.69
± ± ± ±
1.58 1.49 2.17 5.27
6 5 6 7
67.50 140.00 64.16 73.57
± ± ± ±
47.11 66.00 47.21 39.82
131.33 202.00 73.16 277.85
± ± ± ±
50.98 60.15 45.98 22.14*
176.33 300.00 160.66 289.42
± ± ± ±
56.28 41.80 50.02 10.57
16.39 11.58 16.83 14.04
± ± ± ±
2.44 0.80 2.02 1.80
caused by chronic systemic administration of fluoxetine. Previously reported evidence has implicated opioid receptors in the panicolytic-like effect of the same selective serotonin reuptake inhibitor (SSRI). More precisely, the anti-panic effect of chronic fluoxetine treatment in rodents subjected to the ETM was shown to be antagonized by intra-dPAG microinjection the nonselective opioid receptor antagonist naloxone (Roncon et al., 2012). Adding to the relevance of MORs in the anti-escape effect of fluoxetine, the results of experiment 3B showed that intra-dPAG
pre-treatment with a sub-effective dose of DAMGO (0.1 nmol) accelerated the anti-escape effect of this SSRI. Thus, in combination with this selective MOR agonist 10 mg/kg fluoxetine impaired escape expression after only 7 days of continuous systemic administration. This effect was not observed with the lower dose (5 mg/kg) of the SSRI (experiment 3A). Moreover, as shown in experiment 4, although the prolonged administration of fluoxetine (5 mg/kg) for 21 days failed to change the escape expression, the combination of this treatment with a single injection of 0.1 nmol of
Table 2 Latency (mean ± standard error of the mean) in seconds to withdraw from the enclosed arm (avoidance) and from the open arm (escape) of the elevated T-maze (ETM) and total distance travelled in the circular arena (open-field test) by rats tested in experiment 2. *p < 0.05 compared to the physiological saline-treated group *p < 0.05 compared to the saline group, according to repeated measure ANOVA followed by Duncan's post hoc test. Drug treatment
N
Baseline (s)
Vehicle Fluoxetine 5 Fluoxetine 10 Saline DAMGO 0.1 DAMGO 0.3 DAMGO 0.5
11 8 8 5 5 5 4
16.54 13.87 84.37 31.80 32.80 11.60 35.75
± ± ± ± ± ± ±
3.62 1.98 47.11 20.39 17.66 1.88 27.77
Avoidance 1 (s) 57.54 44.12 143.62 81.40 101.60 56.80 40.25
± ± ± ± ± ± ±
26.44 15.56 40.31 54.88 58.00 34.44 27.01
Avoidance 2 (s) 102.18 150.87 264.00 115.00 148.20 92.80 104.25
± ± ± ± ± ± ±
30.47 49.37 23.76 48.56 65.47 38.15 66.91
Escape (s) 9.54 11.33 15.16 6.80 7.86 13.80 16.25
± ± ± ± ± ± ±
0.73 1.10 2.31 0.61 1.51 1.75* 1.68*
Total distance travelled (m) 17.34 15.71 15.53 14.63 14.99 14.93 13.49
± ± ± ± ± ± ±
1.75 1.51 1.39 0.66 2.67 2.16 2.38
Fig. 3. (A) Combined administration of DAMGO (0.1 nmol) and subchronic administration of fluoxetine (5 mg/kg, i.p., daily, 7 days); (B) Anti-escape effect caused by the combined administration of DAMGO (0.1 nmol) and subchronic administration of fluoxetine (10 mg/kg, i.p., daily, 7 days). Columns represent the mean; bars, the SEM. The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 5e7. *p < 0.05 compared to the saline þ vehicle group; #P < 0.05 compared to the saline þ fluoxetine group.
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Fig. 4. Anti-escape effect caused by the combined administration of DAMGO (0.1 nmol) and chronic administration of fluoxetine (Fluo: 5 mg/kg, i.p., daily, 21 days). Columns represent the mean; bars, the SEM. The three escape trials measured in the experimental test were merged, as described in the statistical analysis section. n ¼ 5e7. *p < 0.05 compared to the saline þ vehicle group; #p < 0.05 compared to the saline þ fluoxetine group.
DAMGO in the dPAG, a dose that was shown to be sub-effective in experiment 3, caused a clear panicolytic-like effect. This result provides further indication that chronic treatment with fluoxetine facilitates MOR-mediated neurotransmission in the dPAG. Consistent with these findings, chronic i.p. treatment (14 days) with fluoxetine (6 mg/kg) or imipramine (15 mg/kg) has been shown to increase the density of neurons expressing MOR in forebrain regions, such as the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices, suggesting that repeated administration of these antidepressants improves MOR neurotransmission in these areas (De Gandarias et al., 1998, 1999). It is noteworthy, however, that this effect of antidepressants may be related to the type of drug used and to the region investigated. For instance, an radioautographic study failed to show this facilitatory effect on cortical MORs of rats chronically treated (21 days) with other antidepressants, such as the SSRI paroxetine, the noradrenaline reuptake blocker reboxetine and the MAO inhibitor moclobemide. While paroxetine was shown to increase the density of MORs in the olfactory tubercle in the same study, it also decreased MOR density in thalamic sub-areas (Vilpoux et al., 2002). Recently gathered evidence by our research group has shown that prior administration of the 5-HT1A-R antagonist WAY-100635 in the dPAG blocks the anti-escape effect caused by 21 days of systemic fluoxetine treatment (Zanoveli et al., 2010), as observed here with the intra-dPAG microinjection of CTOP. Most importantly, reported evidence indicates that in the dPAG, MORs and 5-HT1A-Rs act cooperatively in the regulation of escape expression. Thus, while the inhibitory effect observed in the ETM escape task after intra-dPAG injection of the 5-HT1A-R agonist 8-OH-DPAT is blocked by previous local administration of CTOP, blockade of 5-HT1A-R by WAY-100635 reciprocally antagonizes the anti-escape effect of DAMGO when both drugs are microinjected in the dPAG (Roncon et al., 2013). In agreement with our findings, Kishimoto et al. (2001) showed that MORs and the 5-HT1A-Rs act synergistically in rat midbrain neurons, more specifically in the ventrolateral PAG. According to their results, activation of both MORs and 5-HT1A-Rs on GABAergic presynaptic neural terminals inhibited GABA release. The signaling pathways responsible for the presynaptic modulation elicited by these two receptor types share a common intracellular mechanism, the opening of Kþ channels via PTX-sensitive G proteins. Subthreshold DAMGO concentrations increased presynaptic serotonergic inhibition and had a significant inhibitory effect when combined with subthreshold concentrations of serotonin. Taken
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together, these results suggest presynaptic synergism between MORs and 5-HT1A-Rs in the ventrolateral PAG. A wealth of evidence obtained over the last several years indicates that chronic (21 days), but not acute or short-term (3 days), fluoxetine administration (10 mg/kg) not only increases 5-HT release in the dPAG, as measured by in vivo microdialysis (Zanoveli et al., 2010), but also enhances the functional reactivity of 5-HT1A-R in this midbrain area, suggesting that sensitization of these receptors may have occurred (De Bortoli et al., 2006; Zanoveli et al., 2007). As mentioned above, the results of experiment 3B further indicate that chronic fluoxetine administration increases the responsiveness of MORs in the dPAG. Given the evidence that these receptors may work cooperatively, forming heterodimers, whether the facilitatory changes induced by this SSRI in the functioning of these receptors are temporally related and how long they last after drug discontinuation remain to be explored. One intriguing finding of the current study was that avoidance of the open arms was facilitated by DAMGO injection in the dPAG, but only in animals that were treated with fluoxetine for either 7 (10 mg/kg) or 21 (5 mg/kg) days. It is important to say that, in these animals, the combined treatment of fluoxetine and DAMGO also caused an anti-escape effect, as discussed above. As both avoidance and escape latencies were increased in these experiments, a motor deficit may be one possible explanation for the obtained results. However, the different treatments presently used here did not significantly modify the total distance traveled by the animals in the open-field test. This result indicates that the effects of these drugs in the ETM were not due to non-specific alterations in motor function. A possible explanation is that the synergic interaction between 5-HT1A-Rs and MORs may have also influenced the functioning of other 5-HT or opioid receptors in the same brain area. In a recently published paper, De Melo Yamashita et al. (2011) observed that intra-dPAG injection of the 5-HT2C receptor agonist MK-212 facilitated avoidance acquisition in rodents subjected to the ETM, indicating an anxiogenic-like effect, without interfering with escape expression. This anxiogenic-like effect was blocked by local microinjection of the 5-HT2C receptor antagonist SB-242084. Therefore, facilitation of 5-HT2C receptor activity in the dPAG seems to interfere with behaviors associated with anxiety, but not with panic. It is also worthy of note that the activation of kappa receptors in the same midbrain area has anxiogenic consequences (Motta et al., 1995), but there is no evidence to date indicating that such activation would not impact the expression of panic-related defensive behaviors. 5. Conclusion The present work provides pharmacological evidence for the involvement of MORs in the mediation of fluoxetine effects. They also indicate that the use of opioids in combination with antidepressants may be a relevant strategy to augment and hasten the onset of the therapeutic effects of antidepressants. Acknowledgments Pesquisa do This work was supported by Fundaç~ ao de Amparo a ~o Paulo (FAPESP) (grant 2012/03798-0), Conselho Estado de Sa gico (CNPq) Nacional de Desenvolvimento Científico e Tecnolo (grant 470119/2004-7), Fundaç~ ao de Apoio ao Ensino, Pesquisa e ^ncia do HC-FMRP-USP (FAEPA) (grant 1291/97, 355/2000, Assiste ~o 68/2001 and 15/2003), and a Pro-Rectory of the University of Sa Paulo (USP) Research grant (IaPQ2012; NAP-USP-NuPNE-156). Each organization had no further role in the design of the study; in the collection, analysis and interpretation of the data; in the writing of
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the report; or in the decision to submit the paper for publication. C.M. Roncon is the recipient of a post-doctorate fellowship from FAPESP (process 2012/23238-0) and was involved in the design of the study, performed the behavioral and, analyzed the results and wrote the manuscript. R.C. Almada participated in the data analysis. J. G. Maraschin collaborated in the psychopharmacological experiments. N.C. Coimbra, F. G. Graeff, H. Zangrossi Jr., and E. A. Audi conceived the study, contributed to the analysis of data, and drafted and revised the final manuscript. All authors read and approved the final version of the manuscript. References Almada, R.C., Coimbra, N.C., 2015. Recruitment of striatonigral disinhibitory and nigrotectal inhibitory GABAergic pathways during the organization of defensive behaviour by mice in a dangerous environment with the venomous snake Bothrops alternatus (Reptilia, Viperidae). Synapse 69, 299e313. Almada, R.C., Roncon, C.M., Elias-Filho, D.H., Coimbra, N.C., 2015. Endocannabinoid signaling mechanisms in the substantia nigra pars reticulata modulate GABAergic nigro-tectal pathways in mice threatened by urutu-cruzeiro venomous pit viper. Neuroscience 303, 503e514. Amano, K., Tanikawa, T., Iseki, H., Kawabatake, H., Notani, M., Kawamura, H., Kitamura, K., 1978. Single neuron analysis of the human midbrain tegmentum. Rostral mesencephalic reticulotomy for pain relief. Appl. Neuropsychol. 41, 66e78. Bandler, R., Shipley, M.T., 1994. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? TINS 17, 379e389. Coimbra, N.C., De Oliveira, R., Freitas, R.L., Ribeiro, S.J., Borelli, K.G., Pacagnella, R.C., ~o, M.L., 2006. Moreira, J.E., da Silva, L.A., Melo, L.L., Lunardi, L.O., Branda Neuroanatomical approaches of the tectum-reticular pathways and immunohistochemical evidence for serotonin-positive perikarya on neuronal substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear-induced analgesia. Exp. Neurol. 197, 93e112. Deakin, J.F.W., Graeff, F.G., 1991. 5-HT and mechanisms of defence. J. Psychopharmacol. 5, 305e315. De Bortoli, V.C., Nogueira, R.L., Zangrossi Jr., H., 2006. Effects of fluoxetine and buspirone on the panicolytic-like response induced by the activation of 5-HT1A and 5-HT2A receptors in the rat dorsal periaqueductal gray. Psychopharmacology 183, 422e428. De Gandarias, J.M., Echevarria, E., Acebes, I., Silio, M., Casis, L., 1998. Effects of imipramine administration on m-opioid receptor immunostaining in the rat forebrain. Arzneimittelforschung 48, 717e719. De Gandarias, J.M., Echevarría, E., Acebes, I., Abecia, L.C., Casis, O., Casis, L., 1999. Effects of fluoxetine administration on m-opioid receptor immunostaining in the rat forebrain. Brain Res. 817, 236e240. Del-Ben, C.M., Graeff, F.G., 2009. Panic disorder: is the PAG involved? Neural Plast. 2009, 1e9. De Melo Yamashita, P.S., De Bortoli, V.C., Zangrossi Jr., H., 2011. 5-HT2C receptor regulation of defensive responses in the rat dorsal periaqueductal gray. Neuropharmacology 60, 216e222. Eichenberger, G.C.D., Ribeiro, S.J., Osaki, M.Y., Maruoka, R.Y., Resende, G.C.C., ^a, S.A.L., Da Silva, L.A., Coimbra, N.C., 2002. NeuroCastellan-Baldan, L., Corre anatomical and psychopharmacological evidence for interaction between opioid and GABAergic neuronal pathways in the modulation of fear and defense elicited by electrical and chemical stimulation of deep layers of the superior colliculus and dorsal periaqueductal gray matter. Neuropharmacology 42, 48e59. Fanselow, M.S., 1991. The Midbrain Periaqueductal Gray as a Coordinator of Action in Response to Fear and Anxiety. NATO ASI Series, vol. 213, pp. 151e173. Fanselow, M.S., Lester, L.S., 1988. A functional behavioristic approach to aversively motivated behavior: Predatory imminence as a determinant of the topography of defensive behavior. In: Bolles, R.C., Beecher, M.D. (Eds.), Evolution and Learning. Erlbaum, Hillsdale, N. J, pp. 185e211. Graeff, F.G., 1990. Brain defense systems and anxiety. In: Burrows, G.D., Roth, M., Noyers Jr., (Eds.), Handbook of anxiety. Elsevier Science Publishers, Amsterdam, pp. 307e354. Graeff, F.G., 1994. Neuroanatomy and neurotransmitter regulation of defensive behaviors and related emotions in mammals. Braz. J. Med. Biol. Res. 27, 811e829. Graeff, F.G., Netto Ferreira, C., Zangrossi Jr., H., 1998. The elevated T-maze as an experimental model of anxiety. Neurosci. Biobehav. Rev. 23, 237e246. Graeff, F.G., 2004. Serotonin, the periaqueductal gray and panic disorder. Neurosci. Biobehav. Rev. 28, 239e259. Graeff, F.G., Zangrossi Jr., H., 2010. The dual role of serotonin in defense and the mode of action of antidepressants on generalized anxiety and panic disorders.
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