B2-kinin receptors in the dorsal periaqueductal gray are implicated in the panicolytic-like effect of opiorphin

Accepted Manuscript B2-kinin receptors in the dorsal periaqueductal gray are implicated in the panicolytic-like effect of opiorphin

Caio César Sestile, Jhonatan Christian Maraschin, Marcel Pereira Rangel, Rosangela Getirana Santana, Hélio Zangrossi, Frederico Guilherme Graeff, Elisabeth Aparecida Audi PII: DOI: Reference:

S0278-5846(17)30470-0 doi: 10.1016/j.pnpbp.2017.08.003 PNP 9192

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

16 June 2017 3 August 2017 5 August 2017

Please cite this article as: Caio César Sestile, Jhonatan Christian Maraschin, Marcel Pereira Rangel, Rosangela Getirana Santana, Hélio Zangrossi, Frederico Guilherme Graeff, Elisabeth Aparecida Audi , B2-kinin receptors in the dorsal periaqueductal gray are implicated in the panicolytic-like effect of opiorphin. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pnp(2017), doi: 10.1016/j.pnpbp.2017.08.003

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ACCEPTED MANUSCRIPT B2-kinin receptors in the dorsal periaqueductal gray are implicated in the panicolytic-like effect of opiorphin

Caio César Sestile*1 , Jhonatan Christian Maraschin1 , Marcel Pereira Rangel1 , Rosangela Getirana

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Santana2 , Hélio Zangrossi Jr3.4 , Frederico Guilherme Graeff 4 , Elisabeth Aparecida Audi*1 .

Department of Pharmacology and Therapeutics, State University of Maringá (UEM), Maringá-PR, Brazil. 2 Department

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of Statistical, State University of Maringá (UEM), Maringá-PR, Brazil. 3 Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto-SP, Brazil. 4 Institute of Neurosciences and

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Behavior (INeC), Ribeirão Preto, Brazil.

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* Authors for correspondence: Caio C. Sestile or Elisabeth A. Audi, Department of Pharmacology and Therapeutics, State University of Maringá, Av. Colombo 5790, Maringá, PR, 87020-900, Brazil.

E-mail address: [email protected] (C.C. Sestile); [email protected] (E.A. Audi)

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ACCEPTED MANUSCRIPT Abstract

Reported results have shown that the pentapeptide opiorphin inhibits oligopeptidases that degrade brain neuropeptides, and has analgesic and antidepressant effects in experimental animals, without either tolerance or dependency after chronic administration. In a previous study we showed that opiorphin has a panicolytic-like effect in the dorsal periaqueductal gray (dPAG) electrical stimulation test (EST), mediated by the μ-opioid receptor (MOR). This study further analyzes the

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mechanism of opiorphin panicolytic action, using the EST and drug injection inside the dPAG. The obtained results showed that blockade of the 5-HT1A receptors with WAY-100635 did not change

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the escape-impairing effect of opiorphin, and combined injection of sub-effective doses of

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opiorphin and the 5-HT1A-agonist 8-OH-DPAT did not have a significant anti-escape effect. In contrast, the anti-escape effect of opiorphin was antagonized by pretreatment with the kinin B2 receptor blocker HOE-140, and association of sub-effective doses of opiorphin and bradykinin

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caused a significant anti-escape effect. The anti-escape effect of bradykinin was not affected by previous administration of WAY-100635. Therefore, the anti-escape effect of opiorphin in the

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dPAG seems to be mediated by endogenous bradykinin, acting on kinin B2 receptors, which previous results have shown to interact synergistically with MOR in the dPAG to restrain escape in

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two animal models of panic.

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Keywords: opiorphin, bradykinin, panic model, 5-HT1A receptor; kinin B2 receptor.

Chemical compounds: Opiorphin (PubChem CID: 25195667); WAY100635 maleate salt (PubChem CID: 11957721); 8-OH-DPAT hydrobromide (PubChem CID: 6917794); Bradykinin

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(PubChem CID: 439201); HOE-140 (Icatibant) (PubChem CID: 6918173).

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1. INTRODUCTION

Reported results obtained by our research group with escape responses performed by rats in two models of panic (Moreira et al., 2013) – the elevated T- maze (ETM) and electrical stimulation

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test (EST) of dorsal periaqueductal gray (dPAG) – indicate that endogenous opioids inhibit neurons that organize proximal defense in this brain region (Maraschin et al., 2016; Rangel et al., 2014;

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Roncon et al., 2015, 2013). Because panic attacks may be viewed as dysfunctional activation of proximal defense in the dPAG (Deakin and Graeff, 1991; Mobbs et al., 2007; Shuhama et al.,

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2016), these results implicate opioids in the vulnerability to panic attacks that is characteristic of panic disorder (Graeff, 2017, 2004). In this respect Preter and Klein (2008) have suggested that

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panic patients have a deficient opioid system that buffers panic attacks. The above-mentioned studies with rat models of panic have further shown that the anti-

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escape action of opioids in the dPAG was enacted through activation of the μ-opioid receptor (MOR), and that this receptor synergistically interacts with the serotonin 5-HT1A receptor (5HT1AR) (Rangel et al., 2014; Roncon et al., 2013). The latter has been shown to mediate the anti-

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escape effect of chronic, systemically administered fluoxetine (Roncon et al., 2015, 2012). Like with endogenous opioids, deficient 5-HT inhibition of proximal defense in the dPAG has also been

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supposed to underpin the vulnerability to panic attacks of panic patients (Del-Ben et al., 2001; Johnson et al., 2008). As a consequence, the cooperative interaction between 5-HT and endogenous opioids in the dPAG is thought to reconcile the opioid-deficiency and the 5-HT-deficiency

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hypotheses of panic disorder pathophysiology (Graeff, 2017, 2012). Long-term administration of fluoxetine-like antidepressants that inhibit 5-HT membrane reuptake is the first-choice drug treatment of panic disorder. Although quite effective, these agents have drawbacks that prompt the search for new therapeutic medication, among which are the delay of several weeks for the therapeutic action to take place, the sizable portion of non-responders, and several untoward collateral effects (e.g., Bandelow, Baldwin, and Zwanzger 2013). In this regard, the suggested cooperative interaction between 5-HT1AR and MOR in the dPAG for regulating panic attacks led to the proposal that additional opioid medication could enhance the panicolytic action of antidepressants, possibly accelerating the onset of action and making at least some drug-resistant patients responsive (Roncon et al., 2015). However, available agents that directly stimulate MOR, such as morphine and heroin, induce euphoria and are highly addictive (e.g., Berrettini, 2016). 3

ACCEPTED MANUSCRIPT It follows from the above argument that one should look for chemicals that act on MOR indirectly. A promising option is opiorphin, a pentapeptide that was first isolated from human saliva. Opiorphin impairs the degradation of endogenous enkephalins by inhibiting a neutral endopeptidase and an aminopeptidase N (Wisner et al., 2006). As a result, it may enhance the effects of enkephalins, which are mediated by the activation of the δ-opioid receptor and/or MOR (Thanawala et al., 2008). Results obtained in experimental animals have shown that opiorphin has analgesic (Popik et al., 2010; Rougeot et al., 2010; Wisner et al., 2006) and antidepressant (Javelot

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et al., 2010; Yang et al., 2011) effects, and has low toxicity after acute administration (Bogeas et al., 2013). In addition, prolonged administration of opiorphin did not induced tolerance to the analgesic

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effect (Rougeot et al., 2010), as well as physiological (Popik et al., 2010) or behavioral (Popik et al., 2010; Rougeot et al., 2010) dependence. Because oral administration of opiorphin is ineffective,

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analogs that can be given per os are being developed (Poras et al., 2014).

These properties have prompt us to test opiorphin in animal models of panic. The obtained

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results showed that intravenous injection of opiorphin impaired escape performance in both the ETM and the EST. This panicolytic-like effect was also observed following the intra-dPAG

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administration of opiorphin, and local pretreatment with the selective MOR antagonist CTOP abolished the anti-escape effect of intra-dPAG opiorphin in both animal models, as well as antagonized the effect of intravenous opiorphin in the EST. Therefore opiorphin seems to exert a

by endogenous enkephalins.

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panicolytic-like action in the dPAG, mediated by MOR (Maraschin et al., 2016), possibly activated

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While enkephalin mediation is a plausible hypothesis, alternative or complementary mechanisms are possible, since neuropeptides other than enkephalins may be degraded by the oligopeptidases inhibited by opiorphin. In this regard, bradykinin (BK), a nonapeptide isolated from

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the precursor plasm protein kininogen, through the action of proteolytic enzymes (Beraldo and Andrade, 1997; Rocha e Silva et al., 1949), emerged as strong candidate. Not only BK-like immunoreactivity has been described in the dPAG (Perry and Snyder, 1984), but also BK injection in this brain region has been shown to increase the threshold intensity of the electrical current that elicits escape behavior when applied to the rat dPAG. Furthermore, the same panicolytic-like effect was determined by intra-dPAG injection of morphine, and the effect of either morphine and BK was antagonized by pretreatment with intraperitoneally injected naloxone, indicating MOR participation (Burdin et al., 1992). To further explore the seemingly panicolytic action of BK in the dPAG, we have carried out a study with the EST, and the obtained results showed that intra-dPAG injection of BK increased escape threshold in a dose-dependent way. Both the selective kinin B2 receptor (B2R) antagonist 4

ACCEPTED MANUSCRIPT HOE-140

and

the selective MOR antagonist CTOP blocked this panicolytic-like effect.

Reciprocally, the same effect of the selective MOR agonist DAMGO was antagonized by pretreatment with either CTOP or HOE-140, indicating cross-antagonism between MOR and B2R. Importantly, intra-dPAG injection of captopril, a drug that inhibits the enzymatic degradation of BK (Taddei and Bortoloto, 2016), impaired escape in a dose-dependent way, and this effect was blocked by pretreatment with HOE-140, suggesting mediation by endogenous BK (Sestile et al., 2017). Therefore, BK may physiologically restrain panic attacks in the dPAG through MOR/B2R

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stimulation. The present study aimed to further investigate the mechanism of action of opiorphin using

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intra-dPAG injection and the EST. If the panicolytic-like effect of opiorphin were mediated by

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enkephalins through activation of MOR, it should be affected by drugs that act on the 5-HT1AR, as it happens with other MOR agonists and antagonists (Rangel et al., 2014; Roncon et al., 2013). To test this prediction, the first experiment probed whether the selective 5-HT1AR-blocker WAY-

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100635 would antagonize opiorphin, and the second, whether co-administration of sub-effective doses of opiorphin and the selective 5-HT1AR-agonist 8-OH-DPAT would result in a significant

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panicolytic-like effect. Since both gave negative results, the following experiments focused on BK. Thus, in the third experiment, we measured the influence of pretreatment with the selective B2R antagonist HOE-140 on anti-escape action of opiorphin, and in the fourth, we assessed the

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panicolytic-like effect of combined administration of sub-effective doses do BK and opiorphin.

tool.

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2. METHODS

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Finally we checked whether BK itself could interact with the 5-HR1AR using WAY-100635 as a

2.1. Animals

Male Wistar rats (State University of Maringá) weighing 220-250 g were housed in group of five per cage under a 12/12 h light/dark cycle (lights on at 40 lux intensity from 07:00 to 19:00h) at 22o C±1o C with free access to food and water. The experimental procedures were approved by the State University of Maringá Committee of Ethical Conduct in the Use of Animals in Experiments (1121010415/CEUA) and are in accordance with the International Guiding Principles for Biomedical Resarch Involving Animals (WHO, 1985). All efforts were made to minimize the number of animals used and their suffering.

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ACCEPTED MANUSCRIPT 2.2. Drugs Opiorphin

(Bachem,

USA);

Bradykinin

(H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH

acetate salt) (Bachem, USA) and HOE-140 (Arg-Arg-Pro-Hyp-Gly-Thi-Ser-Tic-Oic-Arg) (Sigma, USA)

were

dissolved

in

a

distilled

water.

(±)-8-hydroxy-2-(di-n-propylamino)

tetralin

hydrobromide (8-OHDPAT, Sigma, USA) and WAY-100635 (Sigma, USA) was dissolved in 0.9% sterile saline. All drugs were administered into the dPAG. For injections a needle (0.3 mm outer diameter) was introduced through the guide cannula until its tip was 1.0 mm below the cannula end,

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and a volume of 0.2 µl was infused over 120 s using a 10 µl microsyringe (Hamilton 701-RN) that

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was attached to a microinfusion pump (Insight, Brazil).

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2.3. Apparatus

Escape behavior induced by electrical stimulation of the dPAG was evaluated in a 40 cm diameter circular arena that was surrounded by 40 cm high walls made of transparent Plexiglas. The

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stimulation current (peak to peak) was generated by a sine-wave stimulator and monitored with an oscilloscope (Minipa, Brazil). The brain electrode was connected to the stimulator by means of an

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electromechanical swivel and flexible cable, allowing ample movement of the animal inside the experimental cage.

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2.4. Surgery

The rats were anesthetized with an intramuscular injection of ketamine (75 mg/kg; União

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Química, Brazil) and xylazine (10 mg/kg; Bayer, Brazil), associated with local anesthesia with 2% lidocaine (Hipolabor, Brazil). The animal’s head was fixed in a stereotaxic frame to implant a chemitrode in the dPAG, according to the coordinates of the rat brain atlas (Paxinos and Watson,

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2005). Holding the incisor bar 2.5 mm below the horizontal plane, the chemitrode was introduced 1.9 mm lateral to the lambda, 5.2 mm below the surface of the skull at an angle of 22°. The chemitrode was made of a stainless steel guide cannula (outside diameter 0.6 mm, 0.4 mm inner diameter) glued to an electrode made of stainless steel wire, enamel insulated except at the cross-section of the tip, reaching 1 mm below the lower end of the cannula. The electrode wire used for brain stimulation was connected to a male pin, parallel to the outer end of the cannula, which could be plugged into an amphenol socket at the end of a flexible electrical cable. The surgeries were performed 7 days before the behavioral test.

2.5. Procedure

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ACCEPTED MANUSCRIPT For the electrical stimulation test (EST), animals were placed inside the experimental cage and the escape threshold was determined using electrical stimuli (alternating current, 60 Hz, 10 s) applied through the chemitrode implanted in the dPAG. The inter-stimulus interval was 10 s. The current intensity started at 20 μA and was increased by 4 μA steps until the rat presented running or jumping reactions, characterizing escape behavior. When these responses were observed, the experimenter interrupted the electrical stimulation of the dPAG. The basal escape threshold was defined as the lowest current intensity that evoked escape in three successive trials of electrical

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stimulation. Animals with basal thresholds above 152 μA were excluded from the study. Drug effects were determined as the difference between post- and pretreatment escape thresholds. An

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increase in this value was considered a panicolytic- like effect.

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2.6. Experimental design

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2.6.1. Experiment 1: Combined injection of WAY-100635 and opiorphin Ten minutes after the determination of the basal escape threshold, the animals were intra-

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dPAG injected with WAY-100635 (0.74 nmol) or saline. Ten minutes later, they were micro injected with opiorphin (5.0 nmol) or saline, and after 10 min, the escape threshold was redetermined. Thus, the following groups were formed: saline + saline (n = 6), saline + opiorphin (n =

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5), WAY + saline (n = 5) and WAY + opiorphin (n = 5). The dose of WAY-100635 was selected

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based on a previous report (de Paula Soares and Zangrossi, 2004).

2.6.2. Experiment 2: Association of sub-effective doses of 8-OH-DPAT and opiorphin Ten minutes after the determination of the basal escape threshold, the animals were intra-

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dPAG injected with a sub-effective dose of 8-OH-DPAT (0.8 nmol) or saline, immediately followed, by either a sub-effective dose of opiorphin (2.5 nmol) or saline. Ten minutes later, the escape threshold was re-determined. Thus, the following groups were formed: saline + saline (n = 6), saline + opiorphin (n = 5), DPAT + saline (n = 6) and DPAT + opiorphin (n = 7). The doses of 8-OH-DPAT and opiorphin were selected based on a previous report (Rangel et al., 2014).

2.6.3. Experiment 3: Combined injection of HOE-140 and opiorphin Ten minutes after the determination of the basal escape threshold, the animals were intradPAG injected with HOE-140 (0.04 nmol) or saline. Ten minutes later, they were injected with opiorphin (5.0 nmol) or saline, and after 10 min, the escape threshold was re-determined. Thus, the following groups were formed: saline + saline (n = 6), saline + opiorphin (n = 7), HOE-140 + saline 7

ACCEPTED MANUSCRIPT (n = 5) and HOE-140 + opiorphin (n = 7). The doses of opiorphin and HOE-140 were selected based on a previous results obtained in our lab (Maraschin et al., 2016; Sestile et al., 2017).

2.6.4. Experiment 4: Association of sub-effective doses of opiorphin and BK Ten minutes after the determination of the basal escape threshold, the animals were intradPAG injected with a sub-effective dose of opiorphin (2.5 nmol) or saline, immediately followed, by either a sub-effective dose of BK (1.0 nmol) or saline. Ten minutes later, the escape threshold

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was re-determined. Thus, the following groups were formed: saline + saline (n = 5), saline + BK (n = 5), opiorphin + saline (n = 6) and opiorphin + BK (n = 7). The dose of BK was selected based on

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a previous report (Sestile et al., 2017).

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2.6.5. Experiment 1: Combined injection of WAY-100635 and BK Ten minutes after the determination of the basal escape threshold, the animals were intra-

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dPAG injected with WAY-100635 (0.74 nmol) or saline. Ten minutes later, they were injected with BK (4.0 nmol) or saline, and after 10 min, the escape threshold was re-determined. Thus, the

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following groups were formed: saline + saline (n = 6), saline + BK (n = 6), WAY + saline (n = 6) and WAY + BK (n = 6).

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2.7. Histology

After the experiments, the animals were anesthetized with thiopental (1 ml/kg, i.p., Cristália,

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Brazil) and perfused through the left ventricle of the heart with phosphate buffered saline followed by 10% formalin solution. Afterward, 0.2 μl of methylene blue (2%) was microinjected into the dPAG to mark the drug injection site. Brains were removed from the skull and maintained in 10%

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formalin. Serial 60 mm coronal sections were cut on a cryostat, mounted on gelatin-coated slides, and stained with neutral red. Only animals with electrical stimulation and injection sites located in the dPAG, which comprises the dorsomedial and dorsolateral columns of the PAG, were included in the statistical analysis.

2.8. Statistical analysis Data were tested for homogeneity of variance and, in case of unequal results, were submitted to a logarithmic transformation prior to analysis. Data were analyzed by two-way analyses of variance (ANOVA) followed by Tukey’s post hoc analysis. The effect size of the ANOVA test was measured by the partial eta square (η2 ) calculation. The level of significance was set at p<0.05. All analyses were performed using the Statistical Package for Social Sciences (IBM SPSS®) version 23.0, date Entry (SPSS, Chicago, IL, USA). 8

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3. RESULTS

Figure 1 shows a diagrammatic representation of drug injection sites within the dPAG.

3.1. Experiment 1: No antagonism of the anti-escape effect of opiorphin by WAY-100635

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Fig. 2A shows that intra-dPAG injection of opiorphin (5.0 nmol) increased the escape threshold, and this panicolytic-like effect was not blocked by pretreatment with the selective 5-

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HT1AR-antagonist WAY-100635 (0.74 nmol). Two-way ANOVA showed significant effects of treatment (F(1,17) = 122.83, p<0.0001) and pretreatment x treatment interaction (F (1,17)

=

5.48,

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p<0.05), but not of pretreatment (F(1,17) = 0.02, N.S.). The effect size of the pretreatment x treatment interaction was partial η2 = 0.24. Post hoc analysis showed that the escape thresholds in the WAY +

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opiorphin and saline + opiorphin groups were significantly higher when compared to the saline

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group (p<0.0001).

3.2. Experiment 2: Lack of panicolytic-like effect of the combined injection of subeffective doses of 8-OH-DAPT and Opiorphin

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Fig. 2B shows that a combination of sub-effective doses of the 5-HT1AR-agonist 8-OHDPAT (0.8 nmol) and opiorphin (2.5 nmol) did not increase the escape threshold. Two-way

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ANOVA showed neither significant effects of pretreatment (F (1,20) = 3.81, N.S.) or treatment (F(1,20) = 1.47, N.S.), nor of pretreatment x treatment interaction (F (1,20) = 0.01, N.S.).

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3.3. Experiment 3: Antagonism of the anti-escape effect of Opiorphin by HOE-140 Fig. 3A shows that intra-dPAG injection of opiorphin (5.0 nmol) increased the escape threshold, and this panicolytic-like effect was blocked by pretreatment with the selective B2R antagonist HOE-140 (0.04 nmol). Two-way ANOVA showed significant effects of pretreatment (F(1,21) = 8.60, p<0.01) and treatment (F(1,21) = 14.53, p<0.001), and a pretreatment x treatment interaction (F(1,21)

=

15,34, p<0.001). The effect size of the pretreatment x treatment interaction was

partial η2 = 0.42. Post hoc analysis showed that the escape threshold in the saline + opiorphin group was significantly higher when compared to all the other groups (p<0.001).

3.4. Experiment 4: Panicolytic-like effect of the combined injection of sub-effective doses of Opiorphin and BK 9

ACCEPTED MANUSCRIPT Fig. 3B shows that a combination of sub-effective doses of the opiorphin (2.5 nmol) and BK (1.0 nmol) significantly increased the escape threshold. Two-way ANOVA showed significant effects of pretreatment (F(1,19) = 75.28, p<0.0001) and treatment (F(1,19) = 48.65, p<0.0001), and a pretreatment x treatment interaction (F (1,19) = 30.53, p<0.0001). The effect size of the pretreatment x treatment interection was partial η2 = 0.61. Post hoc analysis showed that the escape threshold in the opiorphin + BK group was significantly higher when compared to all the other groups (p<0.0001).

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3.5. Experiment 5: No antagonism of the anti-escape effect of BK by WAY-100635 Fig. 4 shows that intra-dPAG injection of BK (4.0 nmol) increased the escape threshold, and

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this panicolytic-like effect was not blocked by pretreatment with the selective 5-HT1AR-antagonist WAY-100635 (0.74 nmol). Two-way ANOVA showed significant effects of treatment (F(1,20) = interaction (F(1,20)

=

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82.46, p<0.0001), but not of pretreatment (F(1,20) = 0.60, N.S.) and no pretreatment x treatment 0.87, N.S.). The effect size of the treatment was partial η2 = 0.80. Post hoc

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analysis showed that the escape threshold in the WAY + BK and saline + BK groups were

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significantly higher when compared to saline group (p<0.0001).

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4. DISCUSSION

The results of the first experiment showed that the anti-escape effect of intra-dPAG

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opiorphin in the EST was not antagonized by the 5-HT1AR blocker WAY-100635, and those of the second experiment showed that the combined injection of sub-effective doses of opiorphin and the 5-HT1AR agonist 8-OH-DPAT did not have a significant anti-escape effect. Together, they indicate

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that the 5-HT1AR does not participate in the panicolytic action of opiorphin. Furthermore, they suggest that a primary action on MOR is unlikely, since previous findings have shown that MOR and 5-HT1AR interact synergistically in the dPAG to dampen escape (Rangel et al., 2014; Roncon et al., 2013).

The above results weaken the proposal that opiorphin effects are mediated by endogenous enkephalins (Maraschin et al., 2016). Since the density of the δ-opioid receptor is scant in the dPAG, contrasting with the much higher density of MOR (Mansour et al., 1995), it is likely that enkephalins would act through MOR stimulation. But in that case, they would interact with the 5HT1AR, and, as a consequence, the effect of opiorphin should be antagonized by WAY-100635 and the combined injection of sub-effective doses of opiorphin and 8-OH-DPAT should result in a panicolytic- like effect. Both predictions have not been fulfilled by the present results. 10

ACCEPTED MANUSCRIPT Nevertheless, opiorphin action does seem to involve MOR, as previous results have shown that the MOR blocker CTOP antagonized the anti-escape effect of intra-dPAG opiorphin (Maraschin et al., 2016). This led us to turn to BK as a potential mediator of opiorphin action, since intra-dPAG injection of BK has been shown to have an anti-escape effect in the EST that was antagonized by CTOP (Sestile et al., 2017). The same study also revealed that the selective B2R blocker HOE-140 antagonized the BK effect as well as that of the MOR agonist DAMGO. Furthermore, combined administration of sub-effective doses of BK and DAMGO had a significant

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anti-escape effect. These results indicate a cooperative interaction between MOR and B2R in the dPAG in order to restrain escape. Finally, inhibition of BK degradation with intra-dPAG captopril

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caused an anti-escape effect that was antagonized by HOE-140, pointing to mediation by endogenous BK, which is likely to occur in this brain area (Perry and Snyder, 1984).

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To test the hypothesis of BK mediation, the third experiment of the present study was performed to verify whether HOE-140 would antagonize the anti-escape effect of opiorphin, a

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prediction that was fulfilled by the obtained results. In addition, the fourth experiment has shown that association of sub-effective doses of opiorphin and BK resulted in a significant anti-escape

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effect. Therefore, B2R and MOR seem to interact cooperatively to dampen escape generated in the dPAG, and the anti-escape effect of opiorphin seems to be enacted through endogenous BK. In the same vein, a potentiation of DAMGO effect on sensory neurons by pre-exposure to BK has been

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reported (Berg et al., 2007).

For the last conclusion to hold true, it was necessary to investigate whether the interaction

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between B2R and MOR did not extend to the 5-HT1AR and, indeed, the results of experiment 5 showed that pretreatment with WAY-100635 did not change the anti-escape effect of intra-dPAG BK.

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However, some caveats are due. First, further investigation is needed to explore the possibility of enkephalins to activate the MOR that is associated with the B2R, in which case they could also mediate the actions of opiorphin. Second, the putative role of dynorphin should also be considered, since it has been reported that opiorphin may increase dynorphin and met-enkephalin binding to opioid receptors (Benyhe et al., 2014; Tóth et al., 2012), and that dynorphin A can activate BK receptors (Altier and Zamponi, 2006; Lai et al., 2006). Summarizing, the present and previous results (Maraschin et al., 2016) showed that opiorphin has a panicolytic-like effect in the dPAG mediated by endogenous BK acting through the kinin B2R and the MOR, which interact cooperatively in the dPAG to regulate escape. Since the escape response is a manifestation of proximal defense, and panic attacks may be due to malfunctioning of proximal defense mechanisms in the dPAG (Deakin and Graeff, 1991; Mobbs et 11

ACCEPTED MANUSCRIPT al., 2007; Shuhama et al., 2016), these findings may have translational importance. More specifically, inhibitors of BK degradation, such as orally-active, opiorphin-like drugs (Poras et al., 2014) or kininase II (angiotensin converting enzyme) inhibitors, such as captopril (Cushman and Ondetti, 1991) may be worth investigating as new potential medication of panic disorder. Also worth exploring is the possibility of BK being involved in other therapeutically relevant properties of opiorphin-like drugs, such as the analgesic and antidepressant actions (Javelot et al., 2010; Popik

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et al., 2010; Rougeot et al., 2010; Wisner et al., 2006; Yang et al., 2011)

Financial source: Coordination for the Improvement of Higher Education Personnel

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(CAPES, Brazil) and National Council for Scientific and Technological Development (CNPq,

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Brazil; Grant 466796/2014-5).

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Bandelow, B., Baldwin, D.S., Zwanzger, P., 2013. Pharmacological treatment of panic disorder.

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Benyhe, Z., Toth, G., Wollemann, M., Borsodi, A., Helyes, Z., Rougeot, C., Benyhe, S., 2014. Effects of synthetic analogues of human opiorphin on rat brain opioid receptors. J. Physiol.

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Figures Legend

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Figure 1. Diagrammatic representation of coronal sections of the rat brain (Paxinos and Watson, 2005) showing the location of the injections sites in the dorsal periaqueductal gray matter (dPAG)

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of rats tested in the present study. The number of points is fewer than the total number of rats used because of several overlaps. dlPAG: dorsolateral periaqueductal gray; dmPAG: dorsomedial

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periaqueductal gray; Aq: mesencephalic aqueduct; IA: interaural line.

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Figure 2. Box plot representation (A) intra-dPAG pretreatment with the 5-HT1AR-antagonist WAY100635 (0.74 nmol) did not antagonize the anti-escape effect of the opiorphin (5.0 nmol) and (B)

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the combination of sub effective doses of the opiorphin (2.5 nmol) and the 5-HT1AR-agonist 8-OHDPAT (0.8 nmol) did not increase the escape threshold. The change in threshold (∆) is the difference between escape threshold values (µA) obtained post- and pre-administration of the drug

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Figure 3. Box plot representation (A) intra-dPAG pretreatment with the B2R antagonist HOE 140 (0.04 nmol) antagonized the anti-escape effect of the opiorphin (5.0 nmol) and (B) Anti-escape effect of the combination of sub effective doses of the opiorphin (2.5 nmol) and BK (1.0 nmol). For further information, see the legend of fig. 2. #p<0.05, compared to all the other groups.

Figure 4. Box plot representation intra-dPAG pretreatment with the 5-HT1AR-antagonist WAY100635 (0.74 nmol) did not antagonize the anti-escape effect of the BK (4.0 nmol). For further information, see the legend of fig. 2. *p<0.05, compared to the saline-injected group.

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ACCEPTED MANUSCRIPT Highlights  The panicolytic- like effect of opiorphin in the dPAG is mediated by endogenous bradykinin  B2 receptors mediate this effect  B2 and µ-opioid receptors seem to cooperatively interact for the anti-escape effect of

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