orphanin FQ receptor signaling reverses LPS-induced depressive-like behavior in mice

Accepted Manuscript Title: BLOCKADE OF NOCICEPTIN/ORPHANIN FQ RECEPTOR SIGNALING REVERSES LPS-INDUCED DEPRESSIVE-LIKE BEHAVIOR IN MICE Author: Iris U. Medeiros Chiara Ruzza Laila Asth Remo Guerrini Pedro R.T. Rom˜ao Elaine C. Gavioli Girolamo Calo PII: DOI: Reference:

S0196-9781(15)00162-X http://dx.doi.org/doi:10.1016/j.peptides.2015.05.006 PEP 69480

To appear in:

Peptides

Received date: Revised date: Accepted date:

15-3-2015 18-5-2015 19-5-2015

Please cite this article as: Medeiros IU, Ruzza C, Asth L, Guerrini R, Rom˜ao PRT, Gavioli EC, Calo G, BLOCKADE OF NOCICEPTIN/ORPHANIN FQ RECEPTOR SIGNALING REVERSES LPS-INDUCED DEPRESSIVE-LIKE BEHAVIOR IN MICE, Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

BLOCKADE OF NOCICEPTIN/ORPHANIN FQ RECEPTOR SIGNALING REVERSES LPS-INDUCED DEPRESSIVE-LIKE BEHAVIOR IN MICE

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Iris U. Medeirosa,b, Chiara Ruzza, 1Laila Asth, 3Remo Guerrini, 4Pedro R.T. Romão, 1 Elaine C. Gavioli, 2Girolamo Calo’ 1

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Corresponding author: Elaine C. Gavioli Dept. of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Sen. Salgado Filho, s/n - Lagoa Nova 59072-970 - Natal, RN, Brazil Tel: +55 84 3215-3419 E-mail address: [email protected]

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Dept. of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, RN, Brazil; 2 Dept. of Medical Science, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy; 3 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy; 4 Laboratory of Cellular and Molecular Immunology, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS, Brazil.

HIGHLIGHTS 1 - NOP antagonist did not prevent sickness signs and depressive-like behavior of LPS 2 - NOP antagonists reversed LPS-induced depressive-like behavior in mice 3 - LPS injection evoked similar sickness signs in wild-type and NOP knockout mice 4 - LPS injection elicited depressive effects in wild-type but not in NOP knockout mice 5 - NOP blockade does not affect LPS-induced sickness but reverses depressive behaviors

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Abstract

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Nociceptin/orphanin FQ is the natural ligand of a Gi-protein coupled receptor named NOP. This peptidergic system is involved in the regulation of mood states and inflammatory responses. The present study aimed to investigate the consequences of blocking NOP signaling in lipopolysaccharide (LPS)-induced sickness and depressivelike behaviors in mice. LPS 0.8 mg/kg, ip, significantly induced sickness signs such as weight loss, decrease of water and food intake and depressive-like behavior in the tail suspension test. Nortriptyline (ip, 60 min prior the test) reversed the LPS-induced depressive states. The NOP receptor antagonist SB-612111, 30 min prior LPS, did not modify LPS-induced sickness signs and depressive-like behavior. However, when injected 24 h after LPS, NOP antagonists (UFP-101, icv, and SB-612111, ip) significantly reversed the mood effects of LPS. LPS evoked similar sickness signs and significantly increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) plasma levels 6 h post-injection in wild-type ((NOP(+/+)) and NOP knockout ((NOP(-/-)) mice. However, LPS treatment elicited depressive-like effects in NOP(+/+) but not in NOP(-/) mice. In conclusion, the pharmacological and genetic blockade of NOP signaling does not affect LPS evoked sickness signs while reversing depressive-like behavior.

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Keywords: Nociceptin/orphanin FQ; NOP receptor; lipopolysaccharide-induced depressive; inflammation; depression; tail suspension test.

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1. INTRODUCTION Current preclinical and clinical depression studies have shown that activation of the

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immune system may be involved in major depression. This hypothesis was first proposed based on the high incidence of immunological abnormalities in patients

diagnosed with major depression compared to the general population (Herbert and

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Cohen, 1993) and the development of depression in patients with no prior history of

affective disorders undergoing immunotherapy (Raison et al., 2006). Subsequent studies

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have begun to establish a link between depression and inflammatory conditions such as auto-immune diseases, multiple sclerosis, cardiovascular diseases, diabetes, obesity, as

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well as with others clinical conditions linked with inflammation such as asthma and allergies (Maneeton et al., 2013; Marrie et al., 2009; Brydon et al., 2009; Walker et al., 2011).

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Another interesting fact about the association between depression and inflammation is the similarity of major depression symptomatology with sickness behavior.

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Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and 6 (IL-6) induce behavioral changes such as sleep alterations, cognitive

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dysfunctions, fatigue and anhedonia that are related to alterations in the monoamine systems in brain regions important to mood regulation (for a review see Dantzer et al.,

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2008; Rosenblat et al., 2014).

Considering the role of proinflammatory cytokines on depression, the systemic administration of lipopolysaccharide (LPS), an activator of innate immune system, has been used to trigger depressive-like behavior in rodents. LPS injections in rats and mice induce both peripheral and central inflammation that may result in time related behavioral alterations such increased immobility time in the forced swimming and tail suspension tests (Dunn et al., 2005). Treatment with several classes of antidepressants can revert or prevent this depressive-like behavior (Ohgi et al., 2013; Yirmiya et al., 2001). Nociceptin/orphanin FQ (N/OFQ) is a seventeen-amino acid peptide acting as the endogenous ligand of a G-protein coupled receptor named N/OFQ peptide receptor (NOP) (Reinscheid et al. 1995; Meunier et al., 1995). The peptide precursor of N/OFQ (ppN/OFQ) and NOP receptor are broadly expressed in the central nervous systems as 3 Page 3 of 30

well as in peripheral organs and in the immune system, and the binding of N/OFQ to the NOP receptor modulates a variety of physiological processes (for a review see Lambert, 2008). A growing body of evidence is available in the literature about the role played by N/OFQ-NOP receptor system in inflammation and depression (for a review see Gavioli

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and Romão, 2011; Gavioli and Calo’, 2013). NOP receptor antagonists evoke antidepressant-like effects in different animal models of depression in rats and mice

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(Redrobe et al., 2002; Gavioli et al., 2003; Gavioli et al., 2004; Rizzi et al., 2007; Vitale

et al., 2009; Goeldner et al., 2010). The same molecules when administrated to rodents

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produce beneficial actions in inflammation and sepsis, reducing the mortality in caecal ligation and puncture model of sepsis in rats (Carvalho et al., 2008) and decreasing fecal bleeding in mice dextran sodium sulfate-induced colitis (Alt et al., 2012). In line with

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pharmacological findings, NOP receptor knockout studies corroborated the hypothesis that the N/OFQ-NOP receptor plays an important role in controlling inflammatory

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processes and depression. In fact, the lack of N/OFQ receptor is reported to attenuate the development of colitis in a dextran sodium sulfate murine model (Kato et al., 2005). On the other hand, knocking out the NOP receptor gene induces an antidepressant-like

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phenotype in behavioral despair assays both in mice (Gavioli et al., 2003; Gavioli et al.,

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2004) and rats (Rizzi et al., 2011). Considering this background, the present study aimed to investigate the participation of N/OFQ-NOP receptor in LPS induced sickness

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signs and depressive-like behavior in mice.

2. MATERIAL AND METHODS 2.1. Animals

Experiments were conducted using male Swiss mice breaded at the Federal University of Rio Grande do Norte (Natal, Brazil), CD-1 mice from Harlan (Udine, Italy) and wild type (NOP(+/+)) and NOP knockout (NOP(-/-)) mice, backcrossed on CD-1 strain, breaded at the University of Ferrara (Ferrara, Italy). Mice were 12-16 weeks old (28-35 g) and were housed under standard conditions (22 °C, 12-h light-dark cycle) with food and water ad libitum in plastic cages. Swiss and CD-1 mice were housed in groups of 10-16 per cage (41x34x16 cm) and NOP(+/+) and NOP(-/-) mice were housed in groups of 4 per cage (30x21x14 cm). Mice from Harlan were allowed to acclimatize for at least 7 days before testing. A total number of 129 CD-1 mice, 153 Swiss mice, and 39 and 43 4 Page 4 of 30

NOP(+/+) and NOP(-/-) mice, respectively, were used to develop this study. Experiments performed in Brazil were conducted in accordance with Brazilian law nº 11.794/2008 for care and use of experimental animals. Experiments performed in Italy were performed according to the European (2010/63/EU) and Italian legislation (D. Lgs 26/2014). These protocols were approved by both the Ethic Committee for Animal Use

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of Federal University of Rio Grande do Norte (License Nº 042/2012), and the

University of Ferrara and Italian Ministry of Health (Protocol No. 316/2013-B). This

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study is reported following the ARRIVE guidelines (Kilkenny et al., 2010).

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2.2. Drugs and treatments

LPS (serotype 0111:B4, Sigma, St. Louis, Mo., USA), nortriptyline (Medley

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Pharmaceutical Industry, Campinas, SP, Brazil), UFP-101 ([Nphe1,Arg14,Lys15]N/OFQNH2, synthesized by Prof. Guerrini, Department of Pharmaceutical Sciences, University of Ferrara, Italy), and SB-612111 (Tocris Bioscience, Bristol, UK) were used. LPS and

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nortriptyline were both dissolved in saline solution, while SB-612111 was solubilized in dimethylsulphoxide (DMSO) in a final concentration not exceeding 0.8%. Stock

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solutions of UFP-101 (50 mM) were stored at -20 °C and were diluted to the desired concentration in saline before experiments.

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LPS (0.8 mg/kg) was intraperitoneally (ip) administrated and behavioral and biochemical assays were performed at 6 or 24 h after injection. Nortriptyline (30 mg/kg,

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ip) was injected 60 min before the behavioral assays. The non peptide NOP antagonist SB-612111 (10 mg/kg, ip) was administered following two distinct protocols in separate experimental groups: (1) 30 min before LPS injection, and (2) 30 min before the tail suspension test. The peptide NOP antagonist UFP-101 (10 nmol/2µL) was injected intracerebroventricularly (icv) 5 min before the tail suspension test. Central injections were made at the rate of 1 μl/min through a needle (27 G) protruding 1 mm from the cannula tip. Control groups were treated with their respective vehicles (saline or DMSO 0.8% solutions) following the same schedule described to treatment groups. All drugs injected ip were given in a volume of 10 ml/kg. All drugs were freshly prepared before experiments. The doses of UFP-101 and SB-612111 employed in this study have been previously reported to induce antidepressant-like actions in mice assessed in behavioral despair tests (Gavioli et al., 2003; Gavioli et al., 2004; Rizzi et al., 2007; Goeldner et al., 2010). 5 Page 5 of 30

2.3. Cannula implantation Surgical implantation of cannula in lateral ventricle was conducted in mice anesthetized intraperitoneally with ketamine and xylazine (100 and 10 mg/kg, respectively) and placed in a stereotaxic apparatus. An incision was made in the skin to expose the skull.

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A stainless-steel 8 mm guide cannula (25x0.7 mm) was implanted in lateral ventricle

and was fixed with dental cement. Coordinates toward the bregma were lateral +1.1

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mm, posterior -0.6 mm, and ventral -1.0 mm (Paxinos and Franklin, 2008). To prevent occlusion, a dummy cannula was inserted in the guide cannula. After surgery, pain and

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inflammation were attenuated with subcutaneous diclofenac sodium (10 mg/kg) administration and the animals were allowed to recover for at least 4 days before tests. At the end of the experimental procedures, all animals were euthanized with an

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overdose of sodium thiopental and perfused transcardially with saline solution. The placement of the cannula was verified under a light microscope. Results of animals in

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which the cannula was not correctly placed were excluded for further analysis (less than 10%).

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2.4. LPS-induced sickness signs

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Changes in rectal temperature, body weight, food and water intake following LPS injection were used as parameters to evaluate sickness. Rectal temperature was

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determined using a digital thermometer (MicroTherma 2T Hand Held Thermometer, 2Biological Instruments, Besozzo, Italy) via a lubricated thermistor probe (2-mm diameter) inserted 20 mm into the rectum. The probe was left in place until steady readings were obtained (approximately 20 s). The temperature was measured three times: before LPS injection, and 6 and 24 h after. Body weight and food and water intake were measured before and 24 h after LPS injection.

2.5. Tail suspension test The test was performed during the light cycle (between 09.00 and 13.00) as previously described by Steru et al. (1985). Mice were isolated acoustically and visually and suspended, 50 cm above the floor, by an adhesive tape placed 1 cm from the tip of the tail. The total amount of time each animal remained immobile (i.e. did not struggle) during the 6-min session was recorded (in seconds) as immobility time. The test was performed 24 h after LPS injection. 6 Page 6 of 30

2.6. Open field test Spontaneous locomotor activity of mice was measured using the open field test. The apparatus, made of wood covered with impermeable formica, had a black floor of 40×40 cm and black walls of 40 cm high. The test room had a controlled illumination

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(dimly-light condition). Each mouse was placed in the center of the open field and the

distance travelled (in meters) for 5 min was registered, through automatic observation

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(Anymaze, Stoelting Co., Wood Dale, IL, USA). After the behavioral evaluation of each min after the tail suspension test.

2.7. Serum levels of proinflammatory cytokines

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mouse, the arena was cleaned with 5 % ethanol solution. This test was performed 30

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At 6 h or 24 h after LPS injection, blood was collected by cardiac puncture under isoflurane anesthesia and left 30 min at room temperature. Serum was obtained

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centrifuging the samples for 10 min at 10000 G at 4°C. Serum samples were stored frozen (-80°) until the analysis. TNF-α, IL-1β and IL-6 in serum were measured using a commercially available ELISA kits from Life Technologies Corporation (Monza, Italy)

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and quantified using a microplate reader (450 nm) (Sunrise microplate reader, Tecan

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Trading AG, Männedorf, Switzerland). The results were shown as picograms per milliliter (pg/ml). Assays were sensitive with lower limits of quantification at 3 pg/ml

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for IL-6 and TNF-α, and 7 pg/ml for IL-1β.

2.8. Data analysis

Data are expressed as mean ± sem of n animals. Data were analyzed using unpaired Student’s t-test or two-way ANOVA followed by the LSD post-hoc test (independent factors: LPS injection and NOP antagonist or nortriptyline administration), as specify in figure legends. Differences were considered statistically significant when p≤0.05. For the statistical analysis Statistica software version 7.0 (StatSoft Inc., Tulsa, USA) was used.

3. RESULTS 3.1. LPS-induced sickness signs and depressive-like behavior in mice

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The sickness response of mice challenged by LPS was evaluated at 6 and 24 h after injection. LPS injection in Swiss and CD-1 mice significantly reduced rectal temperature at 6 h, but not at 24 h post-injection (Table 1). Indeed, decrease of food and water intake and loss of body weight was observed at 24 h after LPS-challenge (Table

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1).

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[INSERT TABLE 1]

As shown in Figure 1a, the administration of LPS significantly increased the immobility

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time of Swiss and CD-1 mice in the tail suspension test (Swiss mice: t(21) = 3.20; CD-1 mice: t(27) = 2.36, p<0.05 for both strains). However, LPS treatment did not

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significantly affect mouse locomotion in the open field (p>0.05 for both strains; Fig. 1b).

In a distinct experimental series performed in Swiss mice, the effects of nortriptyline, a

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tricyclic antidepressant, were assessed in LPS-treated mice in the tail suspension test. The administration of LPS significantly increased the immobility time of mice, which

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was reversed by nortriptyline (saline + saline: 75.2 ± 8.4; saline + nortriptyline: 27.2 ±6.2*; LPS + saline: 124.8 ± 8.1*; LPS + nortriptyline: 22.1 + 6.3#; two-way ANOVA,

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factors: F(1,46) = 11.40).

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LSD’s test, *p<0.05 vs. saline + saline, #p<0.05 vs. LPS + saline, interaction between

[INSERT FIGURE 1]

3.2. Effects of NOP receptor antagonists on LPS-induced sickness and depressive-like behavior in mice

The effects of SB-612111 pretreatment on the development of LPS-induced sickness and depressive-like behavior in CD-1 mice are illustrated in Figures 2 and 3. As shown in Figure 2, in this series of experiments LPS injection did not significantly reduce rectal temperature (Fig. 2a) but evoked body weight loss (p<0.05, LPS injection factor: F(1,33) = 30.66; Fig. 2b) and decreased food (p<0.05, LPS injection factor: F(1,33) = 161.72; Fig. 2c) and water intake (p<0.05, LPS injection factor: F(1,33) = 95.67; Fig. 2d) in a period of 24 h. The administration of SB-612111 (10 mg/kg, ip), 30 min prior LPS, did not modify the sickness signs induced by the toxin. 8 Page 8 of 30

[INSERT FIGURE 2]

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As represented in Figure 3, LPS increased the immobility time of vehicle-treated mice

in the tail suspension test (p<0.05, LPS injection factor: F(1,33) = 9.80; Fig. 3). This effect of LPS was slightly attenuated by the pretreatment with SB-612111; however the

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NOP antagonist did not significantly counteract the effects of the toxin in the tail

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suspension test.

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[INSERT FIGURE 3]

The effects of NOP antagonists on the depressive-like behavior induced by LPS are summarized in Figure 4. Two distinct NOP antagonists, UFP-101 (10 nmol, icv) (Fig.

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4a) and SB-612111 (10 mg/kg, ip) (Fig. 4b), injected 5 and 30 min respectively before the tail suspension test, significantly reversed the increase of immobility time induced

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by the toxin administered 24 h before the test. In this series of experiments two-way ANOVA followed by the LSD’s post-hoc test revealed an interaction between the

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independent factors (LPS injection and NOP antagonists) for UFP-101 (p<0.05, F(1,43) =

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4.16; Fig. 4a) and SB-612111 (p<0.05, F(1,58) = 4.73; Fig. 4b) treatments. [INSERT FIGURE 4]

3.3. Phenotype of NOP receptor knockout mice on LPS-induced sickness signs and depressive-like behavior

NOP(+/+) and NOP(-/-) mice were injected with LPS (0.8 mg/kg) and during a 24 h period sickness signs were registered. As shown in Figure 5a, no differences were detected in the basal body temperature between NOP(+/+) and NOP(-/-) mice. However, ip LPS significantly decreased rectal temperature in both NOP(+/+) and NOP(-/-) mice compared to saline (p<0.05, Fig. 5a, LPS injection factor: F(1,36) = 7.40). No significant differences were detected in saline-treated NOP(+/+) and NOP(-/-) mice 9 Page 9 of 30

in terms of body weight, water and food intake (Fig. 5b, 5c, and 5d). Systemic LPS significantly decreased body weight (p<0.05, LPS injection factor: F(1,36) = 19.12; Fig. 5b) in NOP(+/+) and in NOP(-/-) mice compared to saline. LPS also reduced food consumption and water intake in both NOP(+/+) and NOP(-/-) mice but these effects were less attenuated in NOP(-/-) animals (p<0.05 for both parameters, food

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consumption: LPS injection factor: F(1,36) = 104.24, Genotype factor: F(1,36) = 9.52,

Fig. 5c; water intake: LPS injection factor: F(1,36) = 71.87, Genotype factor: F(1,36) =

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16.20, Fig. 5d).

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[INSERT FIGURE 5]

In the tail suspension test, NOP(-/-) treated with saline spent less time immobile compared to NOP(+/+) mice, thus suggesting an antidepressant-like phenotype for mice

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lacking the NOP receptor. In addition, ip LPS significantly increased the immobility time of NOP(+/+) while being completely inactive in NOP(-/-) mice (p<0.05,

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[INSERT FIGURE 6]

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interaction between factors: F(1,36) = 4.31; Fig. 6).

3.4. Serum proinflammatory cytokines levels in LPS-treated NOP receptor knockout mice

Serum levels of TNF-α, IL-6 and IL-1β were evaluated in NOP(+/+) and NOP(-/-) mice collected at 6 and 24 h after LPS treatment (Fig. 7). Only IL-6 levels were detectable in NOP(+/+) and NOP(-/-) mice injected with saline (Fig. 7e). Six hours after the LPS injection both NOP(+/+) and NOP(-/-) mice showed a significant increase in the serum levels of TNF-α (p<0.05, LPS injection factor: F(1,37) = 22.88; Fig.7a), and IL-6 (p<0.05, LPS injection factor: F(1,26) = 22.37; Fig. 7b). At this time the levels of TNF-induced by LPS were similar between genotypes. Regarding the serum levels of IL-1β, LPS injection did not significantly affect this cytokine level in NOP(-/-) and NOP(+/+) (p>0.05 for both factors; Fig. 7c). Twenty-four hours after LPS injection, no significant

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differences were detected between control and LPS-treated mice and between genotypes in the levels of TNF-α (Fig. 7d), IL-6 (Fig. 7e) and IL-1β (Fig. 7f).

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[INSERT FIGURE 7]

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4. DISCUSSION

In the present study we showed that systemic LPS evoked sickness signs, such as

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decreased rectal temperature at 6 h, reduction of food and water intake besides weight loss registered in a period of 24 h after administration. Additionally, a depressive-like

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behavior assessed in the tail suspension test was observed 24 h post-LPS injection in Swiss, CD-1 and NOP(+/+) mice. In line with the literature, low doses of LPS elicit in laboratory animals a transitory but robust inflammatory process characterized by

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sickness behavior, modulated by proinflammatory cytokines (Dantzer, 2006). This behavior peaks after two to six hours, and then gradually vanishes hours after the exposure to the immune challenge. This is followed by the appearance of a depressive-

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like behavior that peaks 24 h after LPS injection and mimics some features of clinical

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depression, including, among others, helplessness and anhedonia (Dantzer et al., 2008). In this study, the LPS-induced depressive-like behavior measured with the tail

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suspension test was significantly reversed by nortriptyline, a tricyclic antidepressant drug. These observations support a robust and replicable inflammation-induced depressive condition evoked by LPS in distinct mice strains and, as previously reported in the literature, standard antidepressants are able to reverse this depressive-like state (Ohgi et al., 2013; Yirmiya et al., 2001). A growing body of preclinical evidence supports an antidepressant-like effect due to the blockade of NOP receptors. These findings have been obtained by using a range of compounds (peptide and non peptide antagonists), different species (rat and mouse) and assays (behavioral despair and chronic mild stress), and knockout studies demonstrating a highly consistent antidepressant-like phenotype of NOP(-/-) mice and rats (for a review see Gavioli and Calo’, 2013). Herein, we have investigated whether NOP receptors signaling might be implicated in the LPS-induced depressive-like behavior in mice. 11 Page 11 of 30

Aiming to further investigate the role of NOP receptor signaling in depressive-like behavior of LPS-treated mice, NOP knockout mice were used. No major differences were detected between NOP(+/+) and NOP(-/-) mice in terms of sickness behavior after LPS administration. A slight but significant difference was registered only in the amount of water intake. Moreover no major differences were recorded between

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NOP(+/+) and NOP(-/-) mice in terms of inflammatory response to LPS, measured as increasing in serum levels of proinflammatory cytokines. These evidences suggest that

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the endogenous N/OFQ-NOP signaling does not play a major role in the modulation of

the inflammatory response after LPS administration, at least for the parameters

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considered in this study. On the contrary, significant differences were displayed in the behavior of NOP(+/+) and NOP(-/-) mice subjected to the tail suspension test 24 h after LPS administration. In fact, NOP(-/-) mice displayed significantly lower immobility

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time in the tail suspension test compared to wild type animals. LPS significantly increased the immobility time of NOP(+/+) but it was completely inactive in NOP(-/-)

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mice. These observations support an antidepressant-like phenotype for LPS-challenged NOP(-/-) mice. The resistance of mice lacking the NOP receptor to the behavioral effects of LPS can be ascribed both to a different responsiveness to the neurobiological

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modifications induced by LPS soon after its administration that lead to the development tail suspension test.

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of depression, or to a different expression of the despaired behaviors in the day of the

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Of note, using NOP(-/-) mice is not possible to discriminate in which of these two processes the NOP receptor is important, since these mice lack the receptor in all the phases of the test. To address this point antagonism studies have been performed using the selective NOP antagonist SB-612111 given before LPS administration and before the tail suspension test. Confirming the data obtained with the genetic block of the receptor, SB-612111 did not change the sickness parameters of mice treated with LPS, demonstrating that the N/OFQ-NOP signaling does not control the sickness induced by LPS. Importantly, no significant differences were recorded in the tail suspension test between mice treated and untreated with SB-612111 before LPS. These results suggest that the N/OFQergic signaling does not play a role in modulating the neural mechanisms that leads to a depressive-like state after LPS administration. On the contrary, when SB-612111 was injected before the tail suspension test it was able to completely block the increase of immobility time induced by LPS, supporting a role of the NOP receptor in the expression of the despaired behavior induced by LPS. The 12 Page 12 of 30

results obtained with the non peptide antagonist SB-612111 were confirmed with the peptide NOP antagonist UFP-101, that given 5 minutes before the tail suspension test, counteracts LPS-induced depressive-like behavior. Thus, knockout studies, completed by antagonism studies, demonstrated the involvement of the N/OFQ-NOP receptor system in the expression rather than development of inflammation-induced depression.

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It is worth noting that in this study the NOP antagonists SB-612111 and UFP-101 given prior the tail suspension test did not significantly changed per se immobility time of

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control mice. Previous data have showed the antidepressant-like effects of both antagonists in the tail suspension test (Gavioli et al., 2004; Rizzi et al., 2007). However,

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the different experimental conditions may explain these discrepant results. In previous studies, to improve response reliability, mice were submitted to an initial 5-min training session of tail suspension 24 h before the test. Due to these experimental differences,

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the immobility time of control mice in the present study is significantly lower (ranging from 60 to 80 s) than in previous studies (ranging from 160 to 180 s). Therefore these

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large differences in baseline immobility time may likely affect the sensitivity of the assay in detecting the antidepressant-like effects of NOP antagonists in control mice. Considering that proinflammatory cytokines are deeply involved in the pathogenesis of

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depression (Duman et al., 1997; Bilbo and Schwarz, 2009), in this study the serum

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levels of TNF-α, IL-6 and IL-1β were assessed at 6 and 24 h post-LPS injection in NOP(+/+) and NOP(-/-) mice. Proinflammatory cytokines involved in the acute phase

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of inflammation are released in a transient way after an immune challenge (Erickson and Banks, 2011; Kang et al., 2011). In agreement with the literature, the present study showed that at 6 h post-LPS IL-6 and TNF-α were significantly increased in NOP(+/+) mice. However, no significant changes in IL-1β levels were found, which is in agreement with published data that showed the time point peak for this cytokine is at 2 h after LPS challenge (Biesmans et al., 2013). In line with the sickness signs, no significant differences were measured between NOP(+/+) and NOP(-/-) mice on the levels of proinflammatory cytokines at 6 h after LPS injection. These observations contribute to the view that the blockade of NOP receptor signaling does not produce major effects on the development of inflammation elicited by LPS. Indeed, 24 h postLPS injection proinflammatory cytokine levels returned to basal values in NOP(+/+) and NOP(-/-) mice. This suggests that the LPS-induced depressive-like phenotype in NOP(+/+) mice is not due to an ongoing inflammatory process. Notably, no significant differences in the serum levels of proinflammatory cytokines were observed between 13 Page 13 of 30

wild-type and NOP knockout mice at 24 h after LPS. Altogether, the antidepressant-like phenotype of NOP(-/-) mice injected with LPS is not due to differences in serum proinflammatory cytokine levels. Further experiments are required to investigate if any difference in these or others proinflammatory cytokines can be observed in brain tissue of NOP(+/+) and NOP(-/-) mice. Moreover, the role of regulatory cytokines such as IL-

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10 should be investigated in order to further understand the participation of cytokines in the antidepressant-like phenotype of NOP(-/-) mice.

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As discussed above, present findings support the view that NOP antagonists reverse, but not prevent, the LPS-induced depressive-like states in mice. Little information is still

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available about the mechanisms by which NOP antagonists could ameliorate this depressive-like behavior. Actually, one important finding comes from Buzas et al. (2002) which reported that LPS, via proinflammatory cytokines (IL-1β and TNF-α), can

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act up-regulating ppN/OFQ mRNA and immunoreactivity to N/OFQ in rat astrocyte cultures. Indeed, the nuclear factor-κB (NF-κB) pathway seems to be involved in LPS

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inducted N/OFQ biosynthesis, since NF-κB inhibitors antagonized the effect of LPS on N/OFQ expression (Buzas et al., 2002). Another evidence comes from mice challenged with the T-cell-activating bacterial superantigen, Staphyloccocal enterotoxin A, that

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increased levels of ppN/OFQ mRNA in the hypothalamus and amygdala (Kawashima et

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al., 2002). Leggett et al. (2009) found that LPS significantly increased ppN/OFQ transcript expression in the hypothalamus 4 h after injection compared to saline.

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Therefore, it is possible that the increased production of N/OFQ induced by immunological stimuli leads to an augmented NOP receptor signaling, thus evoking an excessive

inhibition

on

monoaminergic

neurotransmittion,

with

subsequent

development of the depressive-like states. A growing body of evidence supports an inhibitory role played by NOP activation on monoaminergic neurotransmittion. In fact, the activation of NOP receptors in the locus ceruleus, dorsal raphe nucleus and ventral tegmental area directly inhibits monoaminergic neurons. Additionally, the activation of presynaptic NOP receptors inhibits monoamines release in the prefrontal cortex, amygdala, accumbens nucleus, and hippocampus, which are involved in the control of mood-related behaviors (for a review see Gavioli and Calo’, 2013). In this view, NOP antagonists could promote the antidepressant-like effects in LPS-treated mice by counteracting the activation of NOP receptor signaling evoked by N/OFQ derived from LPS stimulus (see Figure 8).

14 Page 14 of 30

An additional mechanism by which LPS may promote depressive-like effects involves the regulation of the expression of the enzyme indoleamine 2,3 dioxygenase (IDO). Tryptophan is an essential amino acid required for 5-HT synthesis, and metabolized extrahepatically by IDO. This enzyme is highly inducible by proinflammatory cytokines (Babcock and Carlin, 2000), thus reducing tryptophan availability during inflammation

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which impairs serotonergic neurotransmission (Catena-Dell'Osso et al., 2013). In this view, one study performed in T cells activated with the superantigen SEB showed that

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N/OFQ strongly induces IDO expression in these immune cells (Easten et al., 2009).

Therefore, N/OFQ during inflammation could contribute to reduce tryptophan

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availability, thus ultimately facilitating the depressive-like behavior observed in mice 24 h after LPS. This mechanism cannot explain the acute antidepressant effects of NOP antagonists, but could give support to the effects of N/OFQ-NOP receptor signaling in

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mediating the depressive-like states of LPS (see Figure 8).

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[INSERT FIGURE 8]

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5. CONCLUSION

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The present findings suggest that NOP antagonists are able to counteract, but not

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prevent, the LPS-induced depressive-like behaviors. NOP(-/-) mice are insensitive to the depressive-like effects of LPS; however no differences in the development of sickness signs and proinflammatory cytokines levels have been observed between wild type and NOP knockout mice. Ultimately, this study highlights the involvement of the N/OFQ-NOP receptor system in the expression rather than development of inflammation-induced depression.

6. ACKNOWLEDGMENTS This work was supported by funds from the Brazilian National Council Research (grant N°476291/2012-7, N°304453/2012-9, and N°401289/2014-1 to ECG). IUM and LA 15 Page 15 of 30

were

recipient

of

PhD

scholarships

from

Capes

Foundation

(Process

N#99999.002058/2014-06 and 99999.004785/2014-02, respectively) and Brazilian

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National Research Council.

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Paxinos G, Franklin KBJ (2008) The mouse brain in stereotaxic coordinates, 3rd edn. Academic, San Diego. Raison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol. 2006;27:24-31. Redrobe JP, Calo' G, Regoli D, Quirion R. Nociceptin receptor antagonists display antidepressant-like properties in the mouse forced swimming test. Naunyn Schmiedebergs Arch Pharmacol. 2002;365(2):164-7. Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ Jr, Civelli O. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science. 1995;270(5237):792-4. Rizzi A, Gavioli EC, Marzola G, Spagnolo B, Zucchini S, Ciccocioppo R, Trapella C, Regoli D, Calò G. Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vivo studies. J Pharmacol Exp Ther. 2007;321(3):968-74. Rizzi A, Molinari S, Marti M, Marzola G, Calo' G. Nociceptin/orphanin FQ receptor knockout rats: in vitro and in vivo studies. Neuropharmacology. 2011;60(4):572-9. 18 Page 18 of 30

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Vitale G, Ruggieri V, Filaferro M, Frigeri C, Alboni S, Tascedda F, Brunello N, Guerrini R, Cifani C, Massi M.Chronic treatment with the selective NOP receptor antagonist [Nphe 1, Arg 14, Lys 15]N/OFQ-NH 2 (UFP-101) reverses the behavioural and biochemical effects of unpredictable chronic mild stress in rats. Psychopharmacology (Berl). 2009;207(2):173-89.

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Yirmiya R, Pollak Y, Barak O, Avitsur R, Ovadia H, Bette M, Weihe E, Weidenfeld J. Effects of antidepressant drugs on the behavioral and physiological responses to lipopolysaccharide (LPS) in rodents. Neuropsychopharmacology. 2001;24(5):531-44.

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Legends

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Figure 1- Effect of LPS (0.8 mg/kg, ip) or saline on immobility time (a) and distance travelled (b) in Swiss and CD-1 mice in the tail suspension and open-field tests, respectively. Data are the mean ± sem of 11-12 Swiss mice/group and 14-16 CD-1 mice/group.*p<0.05 vs. saline, according to unpaired Student’s t-test.

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Figure 2- Effects of 30 minutes pretreatment with SB-612111 (10 mg/kg, ip) or vehicle on rectal temperature (a), body weight (b), food (c) and water (d) intake in LPS treated mice. SB-612111 was tested in CD-1 mice. Data are the mean ± sem of 8-11 mice/group. *p<0.05 vs. vehicle, two-way ANOVA followed by LSD post-hoc test.

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Figure 3- Effects of 30 minutes pretreatment with SB-612111 (10 mg/kg ip) or vehicle on the immobility time of control and LPS treated mice in the tail suspension test. SB612111 was tested in CD-1 mice. Data are the mean ± SEM of 8-11 animals. *p<0.05 vs. control, two-way ANOVA followed by LSD post-hoc test.

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Figure 4- Effect of icv UFP-101 10 nmol (a) and ip SB-612111 10 mg/kg (b) on the immobility time of control and LPS treated mice in the tail suspension test. Data are the mean ± sem of 9-13 mice/group (a) and 14-17 mice/group (b). UFP-101 was tested in Swiss mice and SB-612111 was tested in CD-1 mice.*p<0.05 vs. control. #p<0.05 vs. saline or vehicle, according to two-way ANOVA followed by LSD post-hoc test.

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Figure 5- Effects of LPS in NOP receptor knockout (NOP(-/-)) and wild type (NOP(+/+)) mice on rectal temperature (a), body weight (b), food (c) and water (d) intake. Data are the mean ± SEM of 9-12 animals. *p<0.05 vs. saline; #p<0.05 vs. LPStreated NOP(+/+), two-way ANOVA followed by LSD post-hoc test. Figure 6- Effects of LPS on the immobility time of NOP receptor knockout (NOP(-/-)) and wild type (NOP(+/+)) mice in the tail suspension test. Data are the mean ± SEM of 9-12 animals. *p<0.05 vs. saline; #p<0.05 vs. NOP(+/+), two-way ANOVA followed by LSD post-hoc test.

Figure 7- Serum concentration of TNF-α (a,d), IL-6 (b,e) and IL-1β (c,d) 6 and 24 h after LPS or saline injection in NOP(+/+) and NOP(-/-) mice. Data are the mean ± SEM of 7-11 animals. *p≤0.05 vs. saline, two-way ANOVA followed by LSD post-hoc test. Cytokines levels below detection limit were assigned as not detected (N.D.). Figure 8- Schematic representation of the mechanism by which N/OFQ-NOP receptor system modulates LPS-induced depressive states. The present figure summarizes the effects of LPS in increasing proinflammatory cytokines expression. These cytokines stimulate ppN/OFQ expression in the brain, thus increasing NOP receptor signaling and ultimately inhibiting 5-HT neurotransmission. IDO expression is also increased by proinflammatory cytokines and N/OFQ; the increased expression of IDO accelerates the tryptophan metabolism, then consequently reducing 5-HT neurotransmitter in the brain. 20 Page 20 of 30

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NOP antagonists block the NOP signaling and reduce the N/OFQ-induced inhibitory effects on 5-HT neurotransmission.

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a.

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Figure 7 6 h after LPS injection b.

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Table 1- Sickness signs assessed at 6 and 24 h after the LPS injection in Swiss and CD-

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Sickness signs

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CD-1 mice Saline LPS

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36.95 ± 0.20 35.89 ± 0.26* 37.91 ± 0.23 37.17 ± 0.18* Rectal temperature 6 h (ºC) 37.54 ± 0.26 37.65 ± 0.51 37.97 ± 0.66 Rectal temperature 24 h (ºC) 37.45 ± 0.14 0.50 ± 0.53 -2.20 ± 0.46* -0.14 ± 0.14 -1.80 ± 0.44* Loss of body weight (g) 6.25 ± 0.00 1.39 ± 0.00* 5.02 ± 0.20 2.81 ± 0.16* Food intake (g) 7.51 ± 0.00 0.77 ± 0.00* 6.22 ± 0.19 3.47 ± 0.26* Water intake (mL) Data are the mean ± sem of n=8-10 Swiss mice/group; n=14-15 CD-1 mice/group. *p<0.05 vs. saline, according to unpaired Student’s t-test.

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