Cooperative regulation of anxiety and panic-related defensive behaviors in the rat periaqueductal grey matter by 5-HT 1A and µ-receptors

Abstract

Previous results with the elevated T-maze (ETM) test indicate that the antipanic action of serotonin (5-HT) in the dorsal periaqueductal grey (dPAG) depends on the activation endogenous opioid peptides. The aim of the present work was to investigate the interaction between opioid- and serotonin-mediated neurotransmission in the modulation of defensive responses in rats submitted to the ETM. The obtained results showed that intra-dPAG administration of morphine significantly increased escape latency, a panicolytic-like effect that was blocked by pre-treatment with intra-dPAG injection of either naloxone or the 5-HT_1A antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate (WAY-100635). In addition, previous administration of naloxone antagonized both the anti-escape and the anti-avoidance (anxiolytic-like) effect of the 5-HT_1A agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), but did not affect the anti-escape effect of the 5-HT_2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Moreover, the combination of sub-effective doses of locally administered 5-HT and morphine significantly impaired ETM escape performance. Finally, the µ-antagonist D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP) blocked the anti-avoidance as well as the anti-escape effect of 8-OHDPAT, and the association of sub-effective doses of the µ-opioid receptor agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate salt (DAMGO) and of 8-OHDPAT had anti-escape and anti-avoidance effects in the ETM. These results suggest a synergic interaction between the 5-HT_1A and the µ-opioid receptor at post-synaptic level on neurons of the dPAG that regulate proximal defense, theoretically related to panic attacks.

Keywords

Serotonin endogenous opioid peptides elevated T-maze periaqueductal grey matter 5-HT1A receptor µ-opioid receptor

Introduction

Considerable experimental evidence points to the participation of the dorsal periaqueductal grey matter (dPAG) in panic disorder and to an inhibitory role of serotonin (5-HT) on neurons of this mesencephalic region that organize proximal defense and trigger panic attacks (Del-Ben and Graeff, 2009; Graeff, 2004; Graeff and Del-Ben, 2008). Another line of evidence indicates that endogenous opioid peptides buffer panic attacks (Preter and Klein, 2008).

In an attempt to reconcile the two approaches, a recent study (Roncon et al., 2012) explored the interaction between 5-HT and endogenous opioid peptides in the dPAG using the elevated T-maze (ETM) test (Graeff et al., 1993; Viana et al., 1994). This apparatus is made of an arm enclosed by walls perpendicular to two open arms, all elevated from the ground. Two tasks are performed in succession by the same rat. The first one consists of inhibitory avoidance of the open arms and the second, one-way escape from one of the open arms. Because inhibitory avoidance is reduced by anxiolytic drugs while escape is impaired by chronic antidepressant administration and facilitated by acute injection of the panicogenic agent CCK, the first task is considered an animal model of generalized anxiety and the escape task, of panic disorder (Graeff et al., 1998; Graeff and Zangrossi, 2010; Pinheiro et al., 2007; Zangrossi et al., 2001). The results obtained by Roncon et al. (2012) showed that the non-selective opioid receptor antagonist naloxone, injected either systemically or inside the dPAG antagonized the anti-escape (panicolytic) effect of chronic administration of fluoxetine in the ETM: also, pre-treatment with naloxone abolished the anti-escape effect of 5-HT itself, both injected into the dPAG. The conclusion was drawn that the inhibitory action of 5-HT on neurons of the dPAG controlling escape was mediated by endogenous opioid peptides.

The present study further explores the interaction between 5-HT and endogenous opioid peptides in the dPAG using the ETM test. In the first set of experiments, we investigated whether intra-dPAG injection of morphine inhibits escape performance, like 5-HT, and if this effect can be blocked either by naloxone or by the selective 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate [WAY-100635]. We also explored whether combined administration of sub-effective doses of 5-HT and morphine into the dPAG has a synergic effect on escape performance.

In the second set of experiments we investigated what subtypes of 5-HT and opioid receptors are involved in escape regulation. Because previously reported results showed that stimulation of either 5-HT_1A or 5-HT_2A receptors in the dPAG has an anti-escape effect (De Bortoli et al., 2006), we initially verified whether pre-treatment with naloxone would antagonize the anti-escape effect of the 5-HT_1A agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide [8-OH-DPAT], and of the preferential 5-HT_2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride [DOI]. Given that the first, but not the second agonist was affected by naloxone, only 5-HT_1A-acting drugs were used in the following experiments.

Although naloxone has higher affinity for µ- than for either δ- or ĸ-opioid receptors (Robson et al., 1983), it is a rather non-selective agent. Therefore, we checked whether µ-opioid receptors are specifically involved in the interaction with 5-HT_1A receptors by using the selective µ-opioid receptor antagonist D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP) (Hawkins et al., 1989) combined with 8-OH-DPAT. In addition, the effect on ETM escape of a combination of sub-effective doses of 8-OH-DPAT and of the selective µ-opioid receptor agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate salt (DAMGO) (Goldstein, 1987) was compared to the effect of each drug given alone.

In every experiment the effects of the different pharmacological treatments on the inhibitory avoidance task in the ETM and on locomotion inside a square arena were also measured.

Methods and materials

Animals

Male Wistar rats (State University of Maringá) weighing 230–300 g were housed in groups of five per cage in Plexiglas-walled cages in a room maintained at 22±1 °C, with an alternating 12 h:12 h light/dark cycle (lights on from 07:00–19:00 hours), and with free access to food and water except during testing. The experimental procedures adopted had been approved by the State University of Maringá Committee of Ethical Conduct in the Use of Animals in Experiments (072/2010-CEAE), and follow the Biomedical Research Involving Animals (CIMS) recommendations (Geneva, 1985).

Drugs

The following drugs were used: morphine sulfate (Sigma, USA); non-selective opioid receptor antagonist, naloxone hydrochloride (Hipolabor, Brazil); 5-HT_1A receptor antagonist, WAY-100635 (Sigma, USA); 5-HT_1A receptor agonist, 8-OHDPAT (Sigma, USA); 5-HT_2A receptor agonist, DOI (RBI, USA); 5-hydroxytryptamine creatinine sulphate (5-HT, Sigma, USA); µ-receptor antagonist, CTOP (Sigma, Switzerland); and µ-receptor agonist, DAMGO (Sigma, Switzerland). All drugs were dissolved in sterile saline and freshly prepared before testing.

Apparatus

The ETM is made of wood and has three arms of equal dimensions (50 cm×12 cm). One arm, enclosed by 40 cm high walls, is perpendicular to two opposed open arms. To avoid falls, the open arms are surrounded by a 1 cm high Plexiglas rim. The whole apparatus is elevated 50 cm above the floor. Locomotion was measured inside a wooden circular arena (70 cm diameter) with 30 cm high walls. Brightness at the level of the maze arms and open-filed centre was 60 lux.

Surgery

Rats were anesthetized with an intramuscular injection of ketamine (60 mg/kg; União Química, Brazil) and xylazine (12 mg/kg; Bayer, Brazil) and fixed to a stereotaxic frame (David Kopf, USA). Before the implant of stainless-steel guide cannula aimed at the dPAG, the animals received local anesthesia with 2% lidocaine (Hipolabor, Brazil). The cannula (12 mm long; outer and inner diameter 0.6 and 0.4 mm, respectively) was implanted following the coordinates from the atlas Paxinos and Watson (2005): posterior = −6.6 mm from bregma, lateral = −2.2 mm, and deep = −3.6 mm, at an angle of 24º 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. To prevent infection, at the end of surgery, all animals were treated with intramuscular injection of 0.1 mL/100 g of a pentabiotic preparation (Fort Dodge; Brazil) and subcutaneously (0.05 mL/100 g) with the anti-inflammatory flunixin meglumine (Banamine; Mantecorp, Brazil). The surgery for guide cannula implantation was performed seven days before the ETM test.

Microinjection of drugs

For drug injection, a needle (0.3 mm outer diameter) was introduced through the guide cannula until its tip was 1.4 mm below the cannula end. A volume of 0.5 µL was injected over a period of 2 min (0.25 μL/min) using a 10 mL microsyringe (Hamilton 701-RN, USA) attached to a microinfusion pump. The displacement of an air bubble inside polyethylene catheter connecting the syringe needle to the intramesencephalic needle was used to monitor the microinjection. The needle was removed 1 min after the injection was finished.

Procedure

Five days after surgery, the animals were gently handled for 5 min and, 24 h later, the animals were again handled for 5 min and pre-exposed to one of the open arms of the ETM for 30 min. A wooden barrier mounted between the central area of the maze and the proximal end of the arm closed the open arm exit. It has been shown that such pre-exposure makes the escape task more sensitive to antipanic drugs, as it shortens the withdrawal latencies from the open arm during the test (Teixeira et al., 2000). The ETM test was performed 24 h later. In experiment 1, rats were pre-treated with an intra-dPAG microinjection of naloxone (0.5 nmol), WAY-100635 (0.37 nmol) or physiological saline 10 min before intra-dPAG injection of morphine (10 nmol) or physiological saline. Thus, the following groups were formed: saline/saline (n = 9), saline/morphine (n=6), WAY-100635/saline (n = 6), WAY-100635/morphine (n=6), naloxone/saline (n = 8), naloxone/morphine (n=5). After 10 min from the last injection, animals were tested in the ETM test as described below. The doses of naloxone and WAY-100635 were chosen based on previous studies of our group with the same test and midbrain area (De Paula Soares and Zangrossi, 2004; Roncon et al., 2012) while that of morphine derived from the results of a previous pilot study.

In experiment 2, we investigated whether the combined injection of sub-effective doses of morphine and 5-HT would alter the behaviors measured in the ETM. To this aim, independent group of rats were pre-treated with an intra-dPAG microinjection of 5-HT (5 nmol) or physiological saline 5 min before intra-dPAG injection of morphine (0.4 nmol) or physiological saline. Thus, the following groups were formed: saline/saline (n=7), 5-HT/saline (n=5), saline/morphine (n=5), 5-HT/morphine (n=6). After 10 min from the last injection, animals were submitted to the ETM test. At the dose presently selected, 5-HT was found to be ineffective in a previous study of our group with the same test and midbrain area (Zanoveli et al., 2003) while that of morphine derived from the results of a previous pilot study.

In order to assess the involvement of 5-HT receptor subtypes in the mediation the behavioral effects found in experiment 2, in experiment 3(A) rats were pre-treated with intra-dPAG administration of naloxone (0.5 nmol) or physiological saline 10 min before local microinjection of 8-OHDPAT (3.2 nmol) or physiological saline. In experiment 3(B), animals were pre-treated with an intra-dPAG microinjection of naloxone (0.5 nmol) or physiological saline 10 min before local microinjection of DOI (16 nmol) or physiological saline. Thus, in experiment 3(A) the following groups were formed: saline/saline (n=9), naloxone/saline (n=6), saline/8-OHDPAT (n=6), naloxone/8-OHDPAT (n=7). In experiment 3(B), the groups were: saline/saline (n=7), naloxone/saline (n=10), saline/DOI (n=7), naloxone/DOI (n=6). After 10 min from the last injection, animals were submitted to the ETM test. The doses of 8-OH-DPAT and DOI were chosen based on their anti-escape effect revealed by previous studies of our group with the same test and midbrain area (De Paula Soares and Zangrossi, 2004; Zanoveli et al., 2003).

In order to assess whether µ-opioid receptors act cooperatively with 5- HT_1A receptors to regulate the behaviors generated in the ETM, in experiment 4(A) rats were pre-treated with an intra-dPAG microinjection of the µ-opioid receptor antagonist CTOP (1.0 nmol) or physiological saline 10 min before intra-dPAG treatment with 8-OH-DPAT (3.2 nmol) or physiological saline. The following groups were formed: saline/saline (n=9), CTOP/saline (n=6), saline/8-OH-DPAT (n=6), CTOP/8-OH-DPAT (n=8). After 10 min from the last injection, animals were submitted to the ETM test.

In experiment 4(B), rats were pre-treated with combined injections into the dPAG of sub-effective doses of 8-OH-DPAT (0.4 nmol) and of the µ-opioid receptor agonist DAMGO (0.1 nmol). The injection of 8-OH-DPAT preceded that of the opioid agonist by 5 min and testing in the ETM was performed 10 min after the last injection. The groups tested were: saline/saline (n=7), 8-OH-DPAT/saline (n=6), saline/DAMGO (n=6), 8-OH-DPAT/DAMGO (n=6). The doses of CTOP and DAMGO were chosen based on the results of previous pilot studies conducted in our lab.

The ETM test was initiated by the inhibitory avoidance task. For this, each animal was placed at the distal end of the enclosed arm of the ETM facing the intersection. The time taken by the rat to leave this arm with four paws was recorded (baseline latency). This measurement was repeated in two subsequent trials (avoidance 1 and 2) at 30 s intervals. Thirty seconds after the last avoidance trial, the rat was placed at the end of the open arm it had been previously exposed to, and the latency to leave this arm with four paws was recorded in three consecutive trials (escape 1, 2, and 3) at 30 s intervals. A cut-off time of 300 s was established for the avoidance and the escape latency. Thirty seconds after being tested in the ETM, the animal was placed inside the circular arena for 5 min to evaluate locomotion. The total distance travelled was analyzed by a video tracking system (Ethovision; Noldus, Holland).

Histology

After testing, animals were anaesthetized with thiopental (1 mL/kg, (i.p.); Cristália, Brazil), and perfused through the left ventricle of the heart with 0.9% saline followed by 10% formalin solution. Afterwards, 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 60-µm midbrain coronal sections were cut on a cryostat, mounted on gelatine-coated slides and stained with neutral red. Only animals with injection sites located inside the dPAG were included in the statistical analysis. Misplacement of the guide-cannula was found in 35% of animals tested.

Statistical analysis

Repeated-measures analysis of variance (RMANOVA) was used to analyze both avoidance and escape data. Pre-treatment and treatment were the independent factors, and trials (baseline, avoidance 1 and 2, and escape 1–3) were the repeated measures. However, as no effect of trials was detected in the escape task, latencies were merged and the data from each rat were analyzed as mean+standard error of the mean (SEM) of the three trials performed. Further analyses of this measure were conducted with a two-factor analysis of variance (MANOVA), pre-treatment and treatment being the independent factors. Locomotion data were analyzed by one-way analysis of variance (ANOVA). When appropriate, the Duncan post hoc test was used. The significance level was set at p<0.05. Statistica Six Sigma (Statsoft) was used for the statistical analysis.

Results

Figure 1 depicts the sites of drug injections into the dPAG of animals tested in the current study.

Experiment 1: Intra-dPAG injection of naloxone or WAY-100635 and the behavioral effects of morphine.

Figure 2 shows that the anti-escape effect of morphine was antagonized by previous intra-dPAG treatment with naloxone or WAY-100635. MANOVA showed a significant effect of the pre-treatment (F_(2,114)=8.39; p<0.01), treatment (F_(1,114)=12.63; p<0.01) and pre-treatment×treatment interaction (F_(2,114)=6.24; p<0.01). The post hoc analysis revealed that morphine significantly increased escape latency and this effect was blocked by either WAY-100635 or naloxone.

Inhibitory avoidance in the ETM or the distance travelled in the circular arena were not affected by any of the drug treatments employed (Table 1).

Experiment 2: Effect of combined intra-dPAG administration of 5-HT and morphine.

Table 1.

Latency (mean±standard error of the mean (SEM)) in seconds to withdrawal from the enclosed arm (inhibitory avoidance) of the elevated T-maze (ETM) and distance travelled in the open-field by rats tested in experiments 1–4.

Drug treatment	n	Baseline	Avoidance 1	Avoidance 2	Distance travelled (m)
Experiment 1
Saline+saline	9	16.44±5.41	57.33±32.42	108.11±37.48	15.53±1.46
Saline+morphine	6	22.50±6.63	107.50±39.71	158.66±45.91	15.44±1.79
WAY100635+saline	6	9.33±6.63	65.00±39.71	70.66±45.91	15.02±1.79
WAY 100635+morphine	6	13.16±6.63	23.33±39.71	57.83±45.91	14.67±1.79
Naloxone+saline	8	16.87±5.74	50.62±34.39	74.50±39.76	17.55±1.55
Naloxone+morphine	5	25.80±7.27	81.20±43.50	107.00±50.29	12.27±1.96
Experiment 2
Saline+saline	7	24.71±9.57	97.57±44.79	114.57±46.19	13.53±1.32
Saline+morphine	5	40.60±11.32	129.20±53.00	193.60±54.66	11.03±1.56
5-HT+saline	5	12.20±11.32	19.80±53.00	56.60±54.66	16.57±1.56
5-HT+morphine	6	36.33±10.33	131.66±48.38	125.50±49.90	11.27±1.42
Experiment 3(A)
Saline+saline	9	13.44±3.24	27.44±5.81	71.56±23.22	16.51±1.49
Saline+8-OH-DPAT	6	13.66±3.97	23.00±7.12	17.50±28.44^a	13.42±1.83
Naloxone+saline	6	20.16±3.97	16.00±7.12	32.83±28.44	16.67±1.83
Naloxone+8-OH-DPAT	7	14.71±3.68	20.85±6.59	62.57±26.33	14.11±1.69
Experiment 3(B)
Saline+saline	7	15.42±11.28	60.57±43.79	78.71±44.78	15.89±1.43
Saline+DOI	7	34.57±5.32	101.57±43.79	144.42±44.78	13.96±1.43
Naloxone+saline	10	22.20±9.44	74.60±36.63	94.30±37.46	16.91±1.20
Naloxone+DOI	6	8.83±12.18	57.16±47.29	150.00±48.36	13.29±1.55
Experiment 4(A)
Saline+saline	9	10.88±2.86	21.00±4.94	53.33±9.98	15.17±1.19
Saline+8-OH-DPAT	6	7.83±3.50	11.33±6.06	10.83±12.22^b	11.88±1.46
CTOP+saline	6	21.00±3.50	34.50±6.06	46.83±12.22	12.57±1.46
CTOP+8-OH-DPAT	8	9.25±3.03	21.25±5.24	39.37±10.58	15.70±1.27
Experiment 4(B)
Saline+saline	7	26.28±12.26	47.42±29.79	133.71±36.25	13.89±2.28
Saline+DAMGO	6	31.33±13.24	78.33±32.17	100.50±39.15	12.74±2.47
8-OH-DPAT+saline	6	28.50±13.24	82.83±32.17	137.16±39.15	14.47±2.47
8-OH-DPAT+DAMGO	6	7.16±13.24	9.16±32.17	10.83±39.15^a	14.32±2.47

5-HT: serotonin; 8-OH-DPAT: (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide ; CTOP: D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN; DAMGO: [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate salt ; DOI: (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride; WAY100635: N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate.

p<0.05 compared to the control group (saline+saline); ^bp<0.05 compared to all groups.

Figure 3 shows the effect caused by the microinjection of sub-effective doses of 5-HT and morphine on escape expression. MANOVA showed significant effects of pre-treatment (F_(1,65)=3.87; p=0.05), treatment (F_(1,65)=14.50; p<0.01) and pre-treatment×treatment interaction (F_(1,65)=5.48; p<0.05). The Duncan test revealed that the group treated with both drugs had significantly longer escape latency than all other groups.

Neither inhibitory avoidance in the ETM nor locomotion in the circular arena differed among the groups tested (Table 1).

Experiment 3(A): Intra-dPAG injection of naloxone and the behavioral effects of 8-OH-DPAT.

As can be seen in Figure 4(a), intra-dPAG injection of 8-OH-DPAT prolonged escape latency and this panicolytic-like effect was antagonized by naloxone. ANOVA showed a significant main effect of pre-treatment (F_(1,80)=14.78 p<0.01), treatment (F_(1,80)=10.27; p<0.01) and pre-treatment×treatment interaction (F_(1,80)=4.22; p<0.05). The post hoc test showed that 8-OH-DPAT significantly increased escape latency compared to all other experimental groups.

Table 1 show that intra-dPAG injection of 8-OH-DPAT impaired inhibitory avoidance acquisition and this effect was counteracted by previous microinjection of naloxone. RMANOVA revealed a significant main effect of trial (F_(2,48)=4.74; p<0.05), but no significant effect of treatment or pre-treatment. However, there was a nearly significant pre-treatment×treatment×trial interaction (F_(2,48)=2.6; p=0.08). Post hoc comparisons showed that 8-OH-DPAT significantly decreased avoidance 2 latency compared to the control, indicating an anxiolytic effect. This effect was counteracted by previous treatment naloxone.

Locomotion in the circular arena did not differ among the groups tested (Table 1).

Experiment 3(B): Intra-dPAG injection of naloxone and the behavioral effects of DOI.

Figure 4(b) shows that differently from the results observed with 8-OH-DPAT, naloxone did not affect the inhibitory effect of DOI on escape performance. There was a significant effect of treatment (F_(1,86)=39.58; p<0.001), but no significant effect of pre-treatment, pre-treatment×treatment or pre-treatment×treatment×trial interaction. The groups treated with saline+DOI or naloxone+DOI had significantly longer escape latency, when compared to the control group.

Neither the inhibitory avoidance in the ETM nor the locomotion in the circular arena was changed by the treatments employed in experiment 3(B) (Table 1).

Experiment 4(A): Intra-dPAG injection of CTOP and the behavioral effects of 8-OH-DPAT.

Figure 5(a) shows that intra-dPAG injection of 8-OH-DPAT inhibited escape expression and this effect was significantly attenuated by previous microinjection of CTOP. There were significant effects of pre-treatment (F_(1,83)=4.73; p<0.05), treatment (F_(1,83)=31.73; p<0.001) and pre-treatment×treatment interaction (F_(1,83)=13.71; p<0.01). Duncan’s post hoc test revealed that 8-OH-DPAT prolonged escape latency when compared to all other groups.

Table 1 shows that 8-OH-DPAT also impaired inhibitory avoidance acquisition and this anxiolytic effect was antagonized by CTOP. RMANOVA revealed significant effects of trial (F_(2,50)=16.94; p<0.05), treatment (F_(1,25)=6.93; p<0.05) and pre-treatment×treatment×trial interaction (F_(2,50)=3.70; p<0.05). The post hoc test showed that animals treated with 8-OH-DPAT had shorter avoidance 2 latency when compared to all other groups.

Locomotion in the circular arena did not differ among the groups tested (Table 1).

Experiment 4(B): Intra-dPAG injection of sub-effective doses of DAMGO and 8-OH-DPAT.

As shown in Figure 5(b), the association of sub-effective doses of 8-OH-DPAT and DAMGO significantly increased escape latency, indicative of a panicolytic-like effect. There were significant effects of pre-treatment (F_(1,71)=17.45; p<0.01), treatment (F_(1,71)=10.07; p<0.01) and pre-treatment×treatment (F_(1,71)=15.19; p<0.01). The association of these two agonists significantly prolonged escape latency in comparison to all other experimental groups.

Table 1 show that the association of 8-OH-DPAT and DAMGO significantly decreased inhibitory avoidance latencies, indicative of an anxiolytic-like effect. RMANOVA revealed significant effects of trial (F_(2,42)=14.89; p<0.001) and trial×treatment (F_(2,42)=4.12; p<0.05). Animals treated with 8-OH-DPAT and DAMGO had shorter avoidance 2 latency when compared to the control group.

Locomotion in the circular arena was not different among the groups tested (Table 1).

Discussion

The results of a preceding study showed that the anti-escape effect of 5-HT in the ETM was antagonized by pre-treatment with the opioid receptor blocker naloxone, both drugs being microinjected into the dPAG. Therefore, the inhibitory action of 5-HT on neurons of the dPAG controlling escape is likely to be mediated by endogenous opioids (Roncon et al., 2012). Data in the literature also show that both 5-HT_1A and 5-HT_2A receptors in the dPAG mediate the panicolytic-like effect in the ETM (De Paula Soares and Zangrossi, 2004; Zanoveli et al., 2003).

Following this line of investigation, the present results showed that previous administration of naloxone into the dPAG antagonized the anti-escape effect of the 5-HT_1A receptor agonist 8-OHDPAT, but not that of the preferential 5-HT_2A agonist DOI, both given intramesencephalically. These results indicate that the 5-HT_1A, but not the 5-HT_2A receptor is involved in the interaction between 5-HT and endogenous opioid peptides in the dPAG neural substrate that regulates escape performance.

As to the question of how 5-HT and opioids interact, the present results showing that pre-treatment with the selective 5-HT_1A antagonist WAY-100635 blocked the anti-escape effect of morphine, rules out a release of endogenous opioids by 5-HT, and points to an interaction between 5-HT and opioid receptors at the post-synaptic level. This hypothesis is further supported by the subsequent results showing that the combined administration of sub-effective doses morphine and 5-HT had a significant anti-escape effect in the ETM. The present results also indicate that the µ-opioid receptor is the best candidate for interacting with the 5-HT_1A receptor, because not only the selective µ-opioid receptor antagonist CTOP has been shown to antagonize the anti-escape effect of 8-OH-DPAT, but also sub-effective doses of both the 5-HT_1A agonist and of the µ-opioid receptor agonist DAMGO were able to significantly increase escape latencies in the ETM.

Overall, the above evidence suggests a synergic interaction between the 5-HT_1A and the µ-opioid receptor at post-synaptic level of neurons of the dPAG that regulate proximal defense, theoretically related to panic attacks (Del-Ben and Graeff, 2009; Graeff, 2004; Graeff and Del-Ben, 2008) and presently measured by the one-escape task performed in the ETM.

Our data also provide evidence that µ-receptor mediated mechanisms take part in the anxiolytic effect observed in the ETM after stimulation of 5-HT_1A receptors of the dPAG (De Paula Soares and Zangrossi, 2004; Zanoveli et al., 2003). More specifically, as seen with the escape response, the effect of 8-OH-DPAT on avoidance acquisition was blocked by both naloxone and CTOP and was facilitated by combined local administration of DAMGO. It should be highlighted, however, that either 5-HT release in the dPAG via chemical stimulation of the dorsal raphe nucleus or local microinjection of this indolamine (Miguel et al., 2010; Pobbe and Zangrossi, 2005) facilitate inhibitory avoidance acquisition, an effect opposed to that caused by 8-OH-DPAT. A recent study (Yamashita et al., 2011) shows that this anxiogenic effect of 5-HT is mediated by the 5-HT_2C receptor and, importantly, that this receptor subtype is not implicated in the anti-escape effect of 5-HT, which is due to the activation of 5- HT_1A and 5-HT_2A receptors. Therefore, the actual relevance of dPAG 5-HT_1A receptors in anxiety regulation remains to be determined.

It is noteworthy that none of the drugs’ effects reported here in the ETM seem to be due to unspecific changes in locomotion as indicated by the lack of alterations in the circular arena. Moreover, although the motor demand for leaving the enclosed or the open arms of the ETM is the same, 8-OH-DPAT shortened avoidance latencies while prolonged escape time.

The morphological basis of the 5-HT-opioid interaction has not yet been demonstrated. Nevertheless, there is reported evidence showing that β-endorphin- and leu-enkephalin-containing perikarya and varicose fibers (Eichenberger et al., 2002; Osaki et al., 2003) are found within both dPAG columns and corpora quadrigemina neurons responsive to aversive stimuli (Coimbra et al., 2006), as well as into the substantia nigra, pars reticulata (Da Silva et al., 2013), a ventral mesencephalic structure known to send inhibitory pathways to the dorsal midbrain (Castellan-Baldan et al., 2006). Since varicose fibers characteristically contain monoamines (Beaudet and Descarries, 1981; Mori et al., 1987) and β-endorphin has high binding affinity for the µ-opioid receptor (Akil et al 1981; Schoffelmeer, et al., 1991), a possibility worth investigating is that of 5-HT and β-endorphin being co-transmitters in the dPAG (Burnstock, 2004). In fact, 5-HT-containing fibers in the central nervous system are rich in varicosities that usually consist in thin and unmyelinated axons with round or flattened dense-cored vesicles (Beaudet and Descarries, 1981), and inside the dPAG there are both serotonin-labeled perikarya (Coimbra et al., 2006) and fibers (Steinbusch, 1981; Takeuchi et al., 1982), as well as endogenous opioid peptide-labelled neuronal bodies and varicose fibers (Eichenberger et al., 2002; Osaki et al., 2003). An interaction between 5-HT and µ-opioid receptors in the PAG is indicated by reported results showing that a lesion of serotonergic fibers significantly reduced DAMGO binding in this midbrain region, without affecting binding of the selective δ-opioid receptor ligand [3H]DPDPE (Allen et al., 1993).

As to clinical implications, the present and previously reported results (Roncon et al., 2012) point to a joint participation of 5-HT and opioids in the pathophysiology of panic disorder (for discussion, see Graeff, 2012). Because of high abuse liability, the use of classical µ-opioid receptor agonists in the treatment of this condition is inadequate. Nevertheless, opioids with relatively low abuse potential, such as methadone and buprenorphine, may be useful as adjunctive therapy in panic patients resistant to antidepressants, especially when the disorder is associated with chronic pain.

Footnotes

Acknowledgements

The authors thank Marcos A Trombelli for technical assistance.

Conflict of interest

The authors declare no conflict of interest.

Funding

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Centro Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP); Fundação de Amparo ao Ensino, Pesquisa e Assistência do Hospital das Clínicas de Ribeirão Preto (FAEPA - HCFMRP - USP).

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