Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41 DOI 10.1007/s00210-014-1055-4
ORIGINAL ARTICLE
Nociceptin/orphanin FQ induces simultaneously anxiolytic and amnesic effects in the mouse elevated T-maze task Laila Asth & Nataly Correia & Bruno Lobão-Soares & Thereza C. Monteiro De Lima & Remo Guerrini & Girolamo Calo’ & Vanessa P. Soares-Rachetti & Elaine C. Gavioli
Received: 24 June 2014 / Accepted: 6 October 2014 / Published online: 16 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Studies have shown a close relationship between anxiety and aversive memory processing, but few animal models are suitable for investigating the effects of a given compound on anxiety and memory simultaneously. A growing body of evidence suggests anxiolytic and amnesic effects of nociceptin/orphanin FQ (N/OFQ). The mouse elevated Tmaze (ETM) has been shown to detect the effects of drugs on anxiety and memory at the same time. In this study, the effects of intracerebroventricular N/OFQ injected before or immediately after training session were assessed in the ETM task. When pretraining injected, N/OFQ 0.1 nmol significantly decreased the latency to enter an open arm in the training session compared to control, which is suggestive of anxiolysis. In addition, N/OFQ (0.1 and 1 nmol) significantly reduced the latency to enter an open arm during the test session compared to control, thus suggesting memory impairments. However, when N/OFQ was administered posttraining, it did not affect memory retrieval. No alterations L. Asth : N. Correia : B. Lobão-Soares : V. P. Soares-Rachetti : E. C. Gavioli (*) Behavioral Pharmacology Laboratory, Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, s/n, Campus Universitário-Lagoa Nova, Natal 59072-970, RN, Brazil e-mail: [email protected] T. C. M. De Lima Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil R. Guerrini Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, 44121 Ferrara, Italy G. Calo’ Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, 44121 Ferrara, Italy
in locomotion were detected in N/OFQ-treated mice in the open field test. In conclusion, these findings are discussed considering the simultaneous anxiolytic and amnesic effects of N/OFQ. Keywords Anxiety . Memory . Nociceptin/orphanin FQ . Elevated T-maze task
Introduction Animal models of anxiety generally are affected by psychobiological processes, such as motor ability, motivation (deprivation of food and/or water), perception (often of painful stimuli), learning, and memory (Handley 1991). It is remarkable that cognitive factors are not usually assessed in the ethologically available animal models of anxiety, such as elevated plus maze and light-dark box. In addition, a wealth of experimental evidence indicates that the brain structures involved in anxiety and in the modulation of memory, particularly emotional memory, overlap extensively (LeDoux 1993). These general observations give support to the relevance of investigating the behavioral effects of drugs on anxiety and memory simultaneously. The elevated T-maze (ETM), a derivation of the elevated plus maze (Pellow et al. 1985), is a useful animal model for testing anxiety and memory simultaneously (Graeff et al. 1993; Asth et al. 2012), and it has been demonstrated that the critical motivational factor in this ethologically based model is the aversive nature of the open arms (Zangrossi and Graeff 1997). It should be mentioned that the ETM test was previously validated for assessing the effects of anxiolytic drugs, such as acute administration of benzodiazepines, buspirone, and ritanserin, besides the chronic administration of antidepressants (Graeff et al. 1998; Teixeira et al. 2000; Ruzza et al. 2012).
34
Aside from the limited number of studies, some reports employed the ETM task as an animal model of memory in rats (Santos et al. 2006; Cruz-Morales et al. 2008; do Maia et al. 2010) and mice (Takahashi et al. 2005; Asth et al. 2012). Despite differences in protocols used, it has been observed that exposure to stress, striatal serotonin modulation (CruzMorales et al. 2008), exposure to ethanol and methylmercury in rats during central nervous system development (do Maia et al. 2010), and microinjections of antagonist 7-chlorokynurenic acid (Santos et al. 2006) impair aversive memory in the rat ETM. In mice, it has been reported that scopolamine evokes amnesic effects that were reversed by the cannabinoid receptor (CB1) antagonist SR141716A (Takahashi et al. 2005). More recently, our research group has shown that a modified version of the mouse ETM task was sensitive to both anxiety- and memory-modulating drugs. The muscarinic M1 receptor antagonist biperiden impaired memory process, while leaving anxiety-like behaviors unchanged (Asth et al. 2012). However, the administration of diazepam, a standard anxiolytic drug, reduced anxiety and at higher doses impaired memory process in the ETM task (Asth et al. 2012). Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide 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). In vivo studies have demonstrated that N/OFQ modulates a variety of biological functions; of particular relevance for the present study are the effects of N/OFQ on anxiety and memory (Sandin et al. 1997; Griebel et al. 1999; Redrobe et al. 2000; Gavioli et al. 2002; Roozendaal et al. 2007). Additionally, NOP receptors are highly expressed in the hippocampus and amygdala (Bunzow et al. 1994; Fukuda et al. 1994; Lachowicz et al. 1995; Neal et al. 1999a, b; Florin et al. 2000; Bridge et al. 2003), which could be the neuroanatomical substrate of the amnesic and anxiolytic-like profile of N/OFQ. In the present study, we evaluated the effects of N/OFQ, on anxiety and memory in the mouse ETM task. As assessed in a variety of animal assays, N/OFQ is known to be involved in the modulation of anxiety and memory process (for review, see Higgins et al. 2002; Gavioli and Calo’ 2006). However, there is no information about a putative interplay between anxiety and memory directly due to the activation of this system. The N/OFQ-NOP receptor system is a candidate target for the development of innovative anxiolytic drugs (Witkin et al. 2014). It is remarkable that using only one behavioral task to investigate the effects of a given compound on anxiety and memory, it will be possible to compare effective doses on each behavior and to discard or accept a relationship between them. These observations could give support to further studies aimed to investigate the brain areas underlying this interplay and to provide further evidence of side effects of compounds targeted to activate this peptidergic system.
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
Materials and methods Animals Male Swiss mice weighting 25–30 g were housed in groups of 15 to 20 per cage (33×40×17 cm) with food and water ad libitum. Animals were maintained under constant temperature (23±1 °C) and under a 12-h light/dark cycle (lights on at 06:00 hours). Behavioral studies were approved by the local Ethics Committee in the Use of Animal of Federal University of Rio Grande do Norte (Protocol No. 040/2009) and strictly followed the Brazilian Law No. 11.794/2008 for the care and use of experimental animals. Drugs and treatment N/OFQ was synthesized according to published methods (Guerrini et al. 1997) by Prof. Remo Guerrini (Department of Biotechnology and Pharmaceutical Chemistry, University of Ferrara, Italy). N/OFQ was solubilized in saline (0.9 % NaCl) and administered intracerebroventricularly (icv) in a volume of 2 μl unilaterally in separate experimental groups at two distinct times: (1) before the training session and (2) immediately after the training session. Saline was injected in control mice. The icv injections were made at the rate of 1 μl/ min through a needle (27 G) protruding 1 mm from the cannula tip. All mice were randomly assigned and they received only one injection accordingly to the pretreatment protocol: before or after the training session. Intracerebroventricular cannula implantation Surgical implantation of the cannula in the lateral ventricle was conducted in mice anesthetized intraperitoneally (ip) with ketamine and xylazine (100 and 10 mg/kg, respectively) and placed with the help of a stereotaxic apparatus. An incision was made in the skin to expose the skull. A stainless-steel 8-mm guide cannula (25×0.7 mm) was implanted in the lateral ventricle and was fixed with dental cement. Coordinates toward the bregma were lateral +1.1 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, the animals were allowed to recover for at least 5 days. Behavioral tests Elevated T-maze task In this study, the ETM was used to assess anxiety and memory features in mice. The experimental protocol was based on the reports of Asth et al. (2012). The ETM was made of wood and had three arms of equal dimensions (30×6 cm). One arm, enclosed by walls 16 cm high, was perpendicular to two
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
opposed arms. A Plexiglas border 0.5 cm high surrounded the open arms. The whole apparatus was elevated 40 cm above the floor. On the training day, each mouse was placed at the distal end of the enclosed arm facing the intersection of the arms and allowed to explore the enclosed arm. The trial ended when the mouse entered in one of the open arms by placing the four paws or remained in the enclosed arm for a maximum of 300 s. In the training session, mice were re-exposed to the ETM as many times as needed to stay 300 s on the enclosed arm (avoidance criterion). The number of trials to reach the avoidance criterion was recorded. Repeated exposure to the ETM apparatus leads to the inhibitory avoidance learning, which is ethologically based on the aversion experienced by these animals when exposed to unprotected spaces (Graeff et al. 1998). Animals which achieved the avoidance criterion in less than three trials were excluded from the study; these were less than 10 %. The latency to enter an open arm was recorded for each trial (avoidance latency). Importantly, the avoidance latency in the first three trials in the training session (i.e., trial 1, trial 2, and trial 3) was used to estimate the anxiety levels experienced by animals. A 30 s of intertrial interval was adopted and, during this time, animals returned to their home cages. Twenty-four hours after training, mice were re-exposed to the enclosed arm during two subsequent trials (i.e., trial 1 and trial 2) in the test session, and the time in which the animal remained in the enclosed arm was recorded and used to assess memory retrieval. The stretch-attend posture during each trial was recorded when a mouse stretched forward and retracted to original position. This behavior reflects the risk assessment of animals in a place and refers to a pattern of responses (scanning, stretch attend, flat back approach) invariably observed in potentially dangerous situations (Griebel et al. 1995). Herein, as presented in Table 1, the frequency rate of stretch-attend posture was calculated for each trial following this formula: frequency rate=number of stretch-attend postures/duration of trial (s). Additionally, considering the variability on the basal expression of stretch-attend posture among groups, the difference between the frequency rate of stretch-attend posture in the test day (trial 1) and trial 3 in the training session was used to statistically compare the effects of N/OFQ treatment (see Figs. 1 and 2). Experiments were performed in a dimly lit and quiet room and with an observer inside the room. All experiments were carried out between 13:00 and 17:00 hours. The apparatus was cleaned with 5 % ethanol solution between subjects.
Open field test Spontaneous locomotor activity of mice was measured using the open field test. During the test, animals were allowed to
35
freely explore the apparatus during 30 min. 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 (dim light condition). Each mouse was placed in the center of the open field, and the distance traveled every 5 min was registered, through automatic observation (ANY-maze, Stoelting, USA), during 30 min. Locomotor activity was recorded between 13:00 and 17:00 hours. After the behavioral evaluation of each mouse, the arena was cleaned with 5 % ethanol solution.
Experimental design Experiment 1: Effects of icv administration of N/OFQ in mice in the ETM task To assess the effect of N/OFQ on anxiety and memory processes in the ETM task, mice were randomly assigned to different treatment groups, which were administered (1) before the training session: N/OFQ (0.1 and 1 nmol, 2 μl, icv) or saline (2 μl, icv) or (2) immediately after the training session: N/OFQ (0.1 and 1 nmol, 2 μl, icv) or saline (2 μl, icv). These doses were selected based on previous studies (Jenck et al. 1997; Goeldner et al. 2008). Each mouse was submitted to the training session of the ETM task 5 min after the icv administration (Gavioli et al. 2002). Twenty-four hours after training, mice were subjected to the test session of the ETM task and the latency to enter in an open arm was recorded.
Experiment 2: Effects of icv administration of N/OFQ on spontaneous locomotion To investigate the effects of N/OFQ on spontaneous locomotor activity, each mouse was submitted to the open field test. Five minutes after N/OFQ icv injection, mice were placed on the center of the arena and they were allowed to explore the apparatus individually during a period of 30 min.
Verification of ICV placement The placement of the cannula was verified prior to data analysis. After completing the test, mice were euthanized with a solution of sodium thiopental (100 mg/kg, ip). Methylene blue dye (2 μl) was icv injected after transcardiac perfusion with saline solution. Mouse brains were removed from the skull to verify the placement of the stainless-steel cannula. Only the data from those animals with dispersion of the dye throughout the ventricles were used; these were more than 95 %.
36
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
Statistics The data herein presented were reported as mean±SEM. Within-group comparisons along trials were made with nonparametric repeated measures ANOVA (Friedmann’s test), whereas the tests of Kruskal-Wallis, followed by the post hoc Dunn’s test, were used to detect significant differences among treatment groups within the same trial. Nonparametric statistical analysis was employed due to the cutoff time of 300 s established for the latency to enter an open arm. The effects of treatment (vehicle and N/OFQ) on the number of trials to reach the avoidance acquisition and the difference in stretch-attend posture between the test day and trial 3 in the training session and on distance moved were analyzed by ANOVA followed by Dunnett’s test. A P value less than 0.05 was considered to be significant. These analyses were performed by Statistica version 5.1 (StatSoft Inc., Tulsa, USA) and INSTAT version 3.06 (GraphPad Software Inc., San Diego, USA).
Results Experiment 1: Effects of icv N/OFQ in mice in the ETM task The effects of treatment (N/OFQ and vehicle pretraining injected) on the latency to enter an open arm in the first three trials of the training session were analyzed by repeated measures ANOVA. A significant increase between the first and the third trials was detected in vehicle-treated mice (Fig. 1; P= 0.01; Fr=8.67). A trend to increase the latency to enter an open arm between the trials in the training session was observed for N/OFQ 1 nmol (Fig. 1; P=0.08; Fr=5.43). Indeed, repeated measures ANOVA showed no significant differences in the latency to enter an open arm between the first and third trials in N/OFQ 0.1 nmol-treated mice (Fig. 1; P>0.05; Fr= 1.09). These findings suggest that spontaneous avoidance acquisition was observed only in the control group. Additionally, a significant decrease in the latency to enter an Table 1 Effects of icv N/OFQ before and immediately after the training session in the ETM task. Frequency rates of stretch-attend posture were assessed during the training and test sessions
Each value represents the mean± SEM of 8–12 animals. Treatment effects on the stretch-attend posture were examined through nonparametric repeated measures ANOVA, followed by Dunn’s test *P0.05, Fr=4.67). The post hoc analysis revealed a significant decrease in this behavior between the first and third trials in control mice, but not in N/OFQ-treated mice (Table 1). Indeed, as shown in Fig. 1, the number of trials to reach the avoidance criterion in the training session was increased by N/OFQ 0.1 nmol compared to control, but it did not reach the statistical significance (Fig. 1; P=0.07, F(2,27)=2.82; one-way ANOVA, Dunnett’s test). This finding supports impairment in learning processing and/or memory acquisition evoked by N/OFQ 0.1 nmol treatment. No significant effects among groups were detected when the difference in the frequency of stretch-attend posture between the test day (trial 1) and trial 3 in the training session was calculated (Fig. 1; P>0.05). Still considering the effects of treatment (N/OFQ and vehicle pretraining injected) on latency to enter an open arm during the test session, ANOVA showed a significant reduction in this parameter for N/OFQ 0.1 and 1 nmol groups compared to control (Fig. 1; trial 1: P=0.001, KW=12.96; trial 2: P=0.011, KW=8.96). Therefore, the pretraining administration of N/OFQ 0.1 and 1 nmol impaired memory retrieval in the mouse ETM task. The effects of N/OFQ injected posttraining were also evaluated in the ETM task. Figure 2 shows the behavior of animals (without pharmacological treatments) during the training session. Repeated measures ANOVA revealed a significant increase in the latency to enter an open arm between the first and third trials in the training session for all groups (Fig. 2; P
ORIGINAL ARTICLE
Nociceptin/orphanin FQ induces simultaneously anxiolytic and amnesic effects in the mouse elevated T-maze task Laila Asth & Nataly Correia & Bruno Lobão-Soares & Thereza C. Monteiro De Lima & Remo Guerrini & Girolamo Calo’ & Vanessa P. Soares-Rachetti & Elaine C. Gavioli
Received: 24 June 2014 / Accepted: 6 October 2014 / Published online: 16 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Studies have shown a close relationship between anxiety and aversive memory processing, but few animal models are suitable for investigating the effects of a given compound on anxiety and memory simultaneously. A growing body of evidence suggests anxiolytic and amnesic effects of nociceptin/orphanin FQ (N/OFQ). The mouse elevated Tmaze (ETM) has been shown to detect the effects of drugs on anxiety and memory at the same time. In this study, the effects of intracerebroventricular N/OFQ injected before or immediately after training session were assessed in the ETM task. When pretraining injected, N/OFQ 0.1 nmol significantly decreased the latency to enter an open arm in the training session compared to control, which is suggestive of anxiolysis. In addition, N/OFQ (0.1 and 1 nmol) significantly reduced the latency to enter an open arm during the test session compared to control, thus suggesting memory impairments. However, when N/OFQ was administered posttraining, it did not affect memory retrieval. No alterations L. Asth : N. Correia : B. Lobão-Soares : V. P. Soares-Rachetti : E. C. Gavioli (*) Behavioral Pharmacology Laboratory, Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, s/n, Campus Universitário-Lagoa Nova, Natal 59072-970, RN, Brazil e-mail: [email protected] T. C. M. De Lima Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil R. Guerrini Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, 44121 Ferrara, Italy G. Calo’ Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, 44121 Ferrara, Italy
in locomotion were detected in N/OFQ-treated mice in the open field test. In conclusion, these findings are discussed considering the simultaneous anxiolytic and amnesic effects of N/OFQ. Keywords Anxiety . Memory . Nociceptin/orphanin FQ . Elevated T-maze task
Introduction Animal models of anxiety generally are affected by psychobiological processes, such as motor ability, motivation (deprivation of food and/or water), perception (often of painful stimuli), learning, and memory (Handley 1991). It is remarkable that cognitive factors are not usually assessed in the ethologically available animal models of anxiety, such as elevated plus maze and light-dark box. In addition, a wealth of experimental evidence indicates that the brain structures involved in anxiety and in the modulation of memory, particularly emotional memory, overlap extensively (LeDoux 1993). These general observations give support to the relevance of investigating the behavioral effects of drugs on anxiety and memory simultaneously. The elevated T-maze (ETM), a derivation of the elevated plus maze (Pellow et al. 1985), is a useful animal model for testing anxiety and memory simultaneously (Graeff et al. 1993; Asth et al. 2012), and it has been demonstrated that the critical motivational factor in this ethologically based model is the aversive nature of the open arms (Zangrossi and Graeff 1997). It should be mentioned that the ETM test was previously validated for assessing the effects of anxiolytic drugs, such as acute administration of benzodiazepines, buspirone, and ritanserin, besides the chronic administration of antidepressants (Graeff et al. 1998; Teixeira et al. 2000; Ruzza et al. 2012).
34
Aside from the limited number of studies, some reports employed the ETM task as an animal model of memory in rats (Santos et al. 2006; Cruz-Morales et al. 2008; do Maia et al. 2010) and mice (Takahashi et al. 2005; Asth et al. 2012). Despite differences in protocols used, it has been observed that exposure to stress, striatal serotonin modulation (CruzMorales et al. 2008), exposure to ethanol and methylmercury in rats during central nervous system development (do Maia et al. 2010), and microinjections of antagonist 7-chlorokynurenic acid (Santos et al. 2006) impair aversive memory in the rat ETM. In mice, it has been reported that scopolamine evokes amnesic effects that were reversed by the cannabinoid receptor (CB1) antagonist SR141716A (Takahashi et al. 2005). More recently, our research group has shown that a modified version of the mouse ETM task was sensitive to both anxiety- and memory-modulating drugs. The muscarinic M1 receptor antagonist biperiden impaired memory process, while leaving anxiety-like behaviors unchanged (Asth et al. 2012). However, the administration of diazepam, a standard anxiolytic drug, reduced anxiety and at higher doses impaired memory process in the ETM task (Asth et al. 2012). Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide 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). In vivo studies have demonstrated that N/OFQ modulates a variety of biological functions; of particular relevance for the present study are the effects of N/OFQ on anxiety and memory (Sandin et al. 1997; Griebel et al. 1999; Redrobe et al. 2000; Gavioli et al. 2002; Roozendaal et al. 2007). Additionally, NOP receptors are highly expressed in the hippocampus and amygdala (Bunzow et al. 1994; Fukuda et al. 1994; Lachowicz et al. 1995; Neal et al. 1999a, b; Florin et al. 2000; Bridge et al. 2003), which could be the neuroanatomical substrate of the amnesic and anxiolytic-like profile of N/OFQ. In the present study, we evaluated the effects of N/OFQ, on anxiety and memory in the mouse ETM task. As assessed in a variety of animal assays, N/OFQ is known to be involved in the modulation of anxiety and memory process (for review, see Higgins et al. 2002; Gavioli and Calo’ 2006). However, there is no information about a putative interplay between anxiety and memory directly due to the activation of this system. The N/OFQ-NOP receptor system is a candidate target for the development of innovative anxiolytic drugs (Witkin et al. 2014). It is remarkable that using only one behavioral task to investigate the effects of a given compound on anxiety and memory, it will be possible to compare effective doses on each behavior and to discard or accept a relationship between them. These observations could give support to further studies aimed to investigate the brain areas underlying this interplay and to provide further evidence of side effects of compounds targeted to activate this peptidergic system.
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
Materials and methods Animals Male Swiss mice weighting 25–30 g were housed in groups of 15 to 20 per cage (33×40×17 cm) with food and water ad libitum. Animals were maintained under constant temperature (23±1 °C) and under a 12-h light/dark cycle (lights on at 06:00 hours). Behavioral studies were approved by the local Ethics Committee in the Use of Animal of Federal University of Rio Grande do Norte (Protocol No. 040/2009) and strictly followed the Brazilian Law No. 11.794/2008 for the care and use of experimental animals. Drugs and treatment N/OFQ was synthesized according to published methods (Guerrini et al. 1997) by Prof. Remo Guerrini (Department of Biotechnology and Pharmaceutical Chemistry, University of Ferrara, Italy). N/OFQ was solubilized in saline (0.9 % NaCl) and administered intracerebroventricularly (icv) in a volume of 2 μl unilaterally in separate experimental groups at two distinct times: (1) before the training session and (2) immediately after the training session. Saline was injected in control mice. The icv injections were made at the rate of 1 μl/ min through a needle (27 G) protruding 1 mm from the cannula tip. All mice were randomly assigned and they received only one injection accordingly to the pretreatment protocol: before or after the training session. Intracerebroventricular cannula implantation Surgical implantation of the cannula in the lateral ventricle was conducted in mice anesthetized intraperitoneally (ip) with ketamine and xylazine (100 and 10 mg/kg, respectively) and placed with the help of a stereotaxic apparatus. An incision was made in the skin to expose the skull. A stainless-steel 8-mm guide cannula (25×0.7 mm) was implanted in the lateral ventricle and was fixed with dental cement. Coordinates toward the bregma were lateral +1.1 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, the animals were allowed to recover for at least 5 days. Behavioral tests Elevated T-maze task In this study, the ETM was used to assess anxiety and memory features in mice. The experimental protocol was based on the reports of Asth et al. (2012). The ETM was made of wood and had three arms of equal dimensions (30×6 cm). One arm, enclosed by walls 16 cm high, was perpendicular to two
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
opposed arms. A Plexiglas border 0.5 cm high surrounded the open arms. The whole apparatus was elevated 40 cm above the floor. On the training day, each mouse was placed at the distal end of the enclosed arm facing the intersection of the arms and allowed to explore the enclosed arm. The trial ended when the mouse entered in one of the open arms by placing the four paws or remained in the enclosed arm for a maximum of 300 s. In the training session, mice were re-exposed to the ETM as many times as needed to stay 300 s on the enclosed arm (avoidance criterion). The number of trials to reach the avoidance criterion was recorded. Repeated exposure to the ETM apparatus leads to the inhibitory avoidance learning, which is ethologically based on the aversion experienced by these animals when exposed to unprotected spaces (Graeff et al. 1998). Animals which achieved the avoidance criterion in less than three trials were excluded from the study; these were less than 10 %. The latency to enter an open arm was recorded for each trial (avoidance latency). Importantly, the avoidance latency in the first three trials in the training session (i.e., trial 1, trial 2, and trial 3) was used to estimate the anxiety levels experienced by animals. A 30 s of intertrial interval was adopted and, during this time, animals returned to their home cages. Twenty-four hours after training, mice were re-exposed to the enclosed arm during two subsequent trials (i.e., trial 1 and trial 2) in the test session, and the time in which the animal remained in the enclosed arm was recorded and used to assess memory retrieval. The stretch-attend posture during each trial was recorded when a mouse stretched forward and retracted to original position. This behavior reflects the risk assessment of animals in a place and refers to a pattern of responses (scanning, stretch attend, flat back approach) invariably observed in potentially dangerous situations (Griebel et al. 1995). Herein, as presented in Table 1, the frequency rate of stretch-attend posture was calculated for each trial following this formula: frequency rate=number of stretch-attend postures/duration of trial (s). Additionally, considering the variability on the basal expression of stretch-attend posture among groups, the difference between the frequency rate of stretch-attend posture in the test day (trial 1) and trial 3 in the training session was used to statistically compare the effects of N/OFQ treatment (see Figs. 1 and 2). Experiments were performed in a dimly lit and quiet room and with an observer inside the room. All experiments were carried out between 13:00 and 17:00 hours. The apparatus was cleaned with 5 % ethanol solution between subjects.
Open field test Spontaneous locomotor activity of mice was measured using the open field test. During the test, animals were allowed to
35
freely explore the apparatus during 30 min. 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 (dim light condition). Each mouse was placed in the center of the open field, and the distance traveled every 5 min was registered, through automatic observation (ANY-maze, Stoelting, USA), during 30 min. Locomotor activity was recorded between 13:00 and 17:00 hours. After the behavioral evaluation of each mouse, the arena was cleaned with 5 % ethanol solution.
Experimental design Experiment 1: Effects of icv administration of N/OFQ in mice in the ETM task To assess the effect of N/OFQ on anxiety and memory processes in the ETM task, mice were randomly assigned to different treatment groups, which were administered (1) before the training session: N/OFQ (0.1 and 1 nmol, 2 μl, icv) or saline (2 μl, icv) or (2) immediately after the training session: N/OFQ (0.1 and 1 nmol, 2 μl, icv) or saline (2 μl, icv). These doses were selected based on previous studies (Jenck et al. 1997; Goeldner et al. 2008). Each mouse was submitted to the training session of the ETM task 5 min after the icv administration (Gavioli et al. 2002). Twenty-four hours after training, mice were subjected to the test session of the ETM task and the latency to enter in an open arm was recorded.
Experiment 2: Effects of icv administration of N/OFQ on spontaneous locomotion To investigate the effects of N/OFQ on spontaneous locomotor activity, each mouse was submitted to the open field test. Five minutes after N/OFQ icv injection, mice were placed on the center of the arena and they were allowed to explore the apparatus individually during a period of 30 min.
Verification of ICV placement The placement of the cannula was verified prior to data analysis. After completing the test, mice were euthanized with a solution of sodium thiopental (100 mg/kg, ip). Methylene blue dye (2 μl) was icv injected after transcardiac perfusion with saline solution. Mouse brains were removed from the skull to verify the placement of the stainless-steel cannula. Only the data from those animals with dispersion of the dye throughout the ventricles were used; these were more than 95 %.
36
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:33–41
Statistics The data herein presented were reported as mean±SEM. Within-group comparisons along trials were made with nonparametric repeated measures ANOVA (Friedmann’s test), whereas the tests of Kruskal-Wallis, followed by the post hoc Dunn’s test, were used to detect significant differences among treatment groups within the same trial. Nonparametric statistical analysis was employed due to the cutoff time of 300 s established for the latency to enter an open arm. The effects of treatment (vehicle and N/OFQ) on the number of trials to reach the avoidance acquisition and the difference in stretch-attend posture between the test day and trial 3 in the training session and on distance moved were analyzed by ANOVA followed by Dunnett’s test. A P value less than 0.05 was considered to be significant. These analyses were performed by Statistica version 5.1 (StatSoft Inc., Tulsa, USA) and INSTAT version 3.06 (GraphPad Software Inc., San Diego, USA).
Results Experiment 1: Effects of icv N/OFQ in mice in the ETM task The effects of treatment (N/OFQ and vehicle pretraining injected) on the latency to enter an open arm in the first three trials of the training session were analyzed by repeated measures ANOVA. A significant increase between the first and the third trials was detected in vehicle-treated mice (Fig. 1; P= 0.01; Fr=8.67). A trend to increase the latency to enter an open arm between the trials in the training session was observed for N/OFQ 1 nmol (Fig. 1; P=0.08; Fr=5.43). Indeed, repeated measures ANOVA showed no significant differences in the latency to enter an open arm between the first and third trials in N/OFQ 0.1 nmol-treated mice (Fig. 1; P>0.05; Fr= 1.09). These findings suggest that spontaneous avoidance acquisition was observed only in the control group. Additionally, a significant decrease in the latency to enter an Table 1 Effects of icv N/OFQ before and immediately after the training session in the ETM task. Frequency rates of stretch-attend posture were assessed during the training and test sessions
Each value represents the mean± SEM of 8–12 animals. Treatment effects on the stretch-attend posture were examined through nonparametric repeated measures ANOVA, followed by Dunn’s test *P0.05, Fr=4.67). The post hoc analysis revealed a significant decrease in this behavior between the first and third trials in control mice, but not in N/OFQ-treated mice (Table 1). Indeed, as shown in Fig. 1, the number of trials to reach the avoidance criterion in the training session was increased by N/OFQ 0.1 nmol compared to control, but it did not reach the statistical significance (Fig. 1; P=0.07, F(2,27)=2.82; one-way ANOVA, Dunnett’s test). This finding supports impairment in learning processing and/or memory acquisition evoked by N/OFQ 0.1 nmol treatment. No significant effects among groups were detected when the difference in the frequency of stretch-attend posture between the test day (trial 1) and trial 3 in the training session was calculated (Fig. 1; P>0.05). Still considering the effects of treatment (N/OFQ and vehicle pretraining injected) on latency to enter an open arm during the test session, ANOVA showed a significant reduction in this parameter for N/OFQ 0.1 and 1 nmol groups compared to control (Fig. 1; trial 1: P=0.001, KW=12.96; trial 2: P=0.011, KW=8.96). Therefore, the pretraining administration of N/OFQ 0.1 and 1 nmol impaired memory retrieval in the mouse ETM task. The effects of N/OFQ injected posttraining were also evaluated in the ETM task. Figure 2 shows the behavior of animals (without pharmacological treatments) during the training session. Repeated measures ANOVA revealed a significant increase in the latency to enter an open arm between the first and third trials in the training session for all groups (Fig. 2; P
