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The light switch-off response as a putative rodent test of innate fear
Neuroscience 334 (2016) 160–165
THE LIGHT SWITCH-OFF RESPONSE AS A PUTATIVE RODENT TEST OF INNATE FEAR VIVIANE M. SAITO * AND MARCUS L. BRANDA˜O
also being considered a controversial issue. The use of experimental animals has been based on factual and moral assumptions, in which it is thought that animal models are reliable to predict results that will be obtained in human studies and that are morally justifiable to avoid atrocities in human subjects, as it happened in the II World War (Ferdowsian and Gluck, 2015). One of the drawbacks pointed is that behavioral tests often present limited explanatory power, since they measure behavior as an indicator, in an attempt to approach the functional role of the phenomena observed. Recent worldwide discussion on the ethical issues in animal experimentation (Nordgren, 2002; Akhtar, 2015; Ferdowsian and Gluck, 2015) instigates the refinement for methods used in neurobiology of behaviors as fear and anxiety. The concept of fear and anxiety is in itself a matter of debate (LeDoux, 2014; Perusini and Fanselow, 2015), and more so when both concepts are transposed to animal behavior. One of the most reliable tests used to predict the anxiolytic- and anxiogenic-like effects of drugs in rodents is the light/dark test, a proposed ‘‘exploratory” animal behavior model for the anxiolytic action of benzodiazepines (Crawley and Goodwin, 1980). The main parameters to assess the anxiolytic profile of drug treatment are the number of transitions between the two compartments, the latency time for the first passage from the light compartment to the dark one, the movement in each compartment, and the time spent in each compartment. Transitions in this test are considered an index of activity/exploration whereas the time spent in each compartment reflects aversion/attraction (Pitsikas et al., 2008; Bourin, 2015). Although this test exploits the differences in behavior seen in dark and lit areas, its rationale remains on the natural conflict between exploration and avoidance that arises when rodents are exposed to novel environments (Crawley and Goodwin, 1980). Our group has developed an animal test focused on the unconditional fear/anxiety paradigm, continuing the first study by Reis et al. (2004). The Light Switch-Off Test (LSOT) is based on the innate motivation to cease an aversive stimulus (bright light). Light by itself is a complex stimulus with both luminance and thermal components (Burns and Webb, 1994). In our protocol, the luminous stimulus per se evokes the light switch-off response (SOR), which is expressed when the rat crosses from one side to the other in a shuttle box. There is no need for previous conditioning or foot shocks. Since the animal emits a response to reduce its exposure to that stimulus,
Laboratory of Neuropsychopharmacology, FFCLRP, Sa˜o Paulo University, Campus USP, Ribeira˜o Preto, Sa˜o Paulo 14040-901, Brazil Instituto de Neurocieˆncias e Comportamento, Avenida do Cafe´, 2450, Ribeira˜o Preto, SP 14050-000, Brazil
Abstract—Recent discussions on the ethics in animal experimentation instigate the refinement of methods used in Behavioral Neuroscience, particularly regarding fear/anxiety paradigms. We propose the Light Switch-Off Test (LSOT), based on the innate motivation to cease an aversive stimulus (bright light), displayed naturally by rodents in their habitat. Forty-six male adult Wistar rats were allocated into independent groups: control, diazepam at 1 or 2 mg/kg, and meta-Chlorophenylpiperazine (mCPP) at 0.5 or 1 mg/kg. The experimental box has two square compartments, separated by an acrylic portal. In each side of the box, there is a 40-W incandescent light bulb. After a habituation period in the box, 40 light stimuli (trials lasting up to 20 s each) are emitted at random intervals. By crossing compartments during the lighted period, the rat could switch-off the stimulus. Parameters observed are the number of switch-off responses (SORs), latency of SOR and intertrial locomotion. The SOR frequency was higher in rats treated with mCPP at 1 mg/kg, an anxiogenic drug, while diazepam at the doses used in this study did not produce effects. Animals exposed solely to the box for the length of the test did not respond in a false positive way. Therefore, the SOR represents a good index to measure the innate rodent fear of bright-lighten areas, once they react quickly in order to turn off the stimulus. Among its many advantages, the LSOT is a simple, replicable, non-invasive and minimally stressful procedure, since it does not expose animals to excessively aversive stimulus. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: animal test, behavior, fear, rat, diazepam, mCPP.
INTRODUCTION Animal models are widely used to model clinical conditions for basic research (Sousa et al., 2006) though *Correspondence to: V. M. Saito, Laboratory of Neuropsychopharmacology, FFCLRP, Sa˜o Paulo University, Campus USP, Ribeira˜o Preto, Sa˜o Paulo 14040-901, Brazil. Fax: +55-16-33154830. E-mail address: [email protected] (V. M. Saito). Abbreviations: 5-HT, serotonin; ANOVA, analysis of variance; LSOT, Light Switch-Off Test; mCPP, meta-Chlorophenylpiperazine; SOR, switch-off response. http://dx.doi.org/10.1016/j.neuroscience.2016.07.044 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 160
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the bright light used in this protocol apparently served as an aversive stimulus. The response to light is naturally occurring and belongs to the ethological repertoire of rodents in their habitat. In the experimental setup, lever pressing was readily acquired and maintained when each press turned off a lamp for 1 min (Keller, 1941). Other study showed that the same learned response ranged in an inverted U-shaped function of light intensity, with intermediate intensities eliciting the highest response rates (Kaplan et al., 1965). The importance of vision is highlighted in the aversion of open spaces that rodents display when submitted to the elevated plus maze, a traditional unconditioned model for anxiety- and conflict-like behaviors (Garcia et al., 2005). In settings where auditory and olfactory stimuli are controlled, aversion is triggered by the light penetration and image formation in the retina (Morato, 2006). One of the arguments that support the intense light as an aversive stimulus to albino rats is the lack of pigment in their iris and choroid, which reduces their vision adaptation and predisposes to visual damage (Stryjek et al., 2013). Although there is a body of evidence indicating that light is an aversive stimulus by itself (Stern and Laties, 1989, 1998; Garcia et al., 2005), no standardized test protocol has been developed using only light as aversive stimulus to trigger an innate response from the rat. Usually, light is used as conditioned cue that precedes the aversive/rewarding stimulus in other protocols (Cassaday and Thur, 2015; Pezze et al., 2016). Here, we present the protocol that has been used and an initial screening of the effects of two drugs in the test: diazepam (a well-known benzodiazepine used as anxiolytic in clinical practice) and meta-Chlorophenylpiperazine (mCPP), a serotoninergic drug with anxiogenic properties, pharmacologically classified as a non-specific agonist for serotonin (5-HT) receptors (Kennett et al., 1989; Kahn and Wetzler, 1991).
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1 or 2 mg/kg. These doses were used based on previous studies in the elevated plus maze that did not induce sedation and motor impairment. mCPP (RBI, MA, USA) was also dissolved in physiological saline solution and delivered i.p. 15 min before the test, at the doses of 0.5 or 1 mg/kg, based on the minimal dose needed for proaversive effects obtained by Reimer et al. (2015, unpublished data). The control group received i.p. treatment with physiological saline. A group of animals tested in the chamber without any stimulus (‘‘Dark only” group) did not receive any treatment. Apparatus The experimental chamber consisted of a shuttle box comprising two compartments measuring 30 25 25 cm (Insight, Brazil). The side and back walls of the chamber were constructed of black Plexiglas and the ceiling and front door were made of transparent Plexiglas. The chamber was divided by an opaque acrylic portal to allow free exploration. A grid floor comprised 15 stainless steel rods with 2.0 mm diameter, spaced 1.2 mm apart. Two 40 W light bulbs were centered on each side of the rear of the chamber, 12 cm from the floor (Fig. 1A). The light was turned on and off noiselessly. The software and an appropriate interface connected to a PC provided by the manufacturer of the equipment (Esquiva Ativa; Insight, Brazil) allowed for recording and analysis of the frequencies and latency of escape responses as well as the intertrial locomotor activity.
EXPERIMENTAL PROCEDURES Animals A total of 46 male adult Wistar rats (weighting between 260 and 280 g) were used in this study. Animals were housed in collective cages in the colony room (12 h light–dark cycle in a temperature controlled, ventilated room). The experiments were carried out during the light phase of the cycle, between 08:00 a.m. and 17:00 p.m. All procedures followed the guidelines on the ethical use of animals by the Brazilian Society of Neuroscience and Behavior (SBNeC) which follows the National Institutes of Health (NIH) guide for the care and use of laboratory animals. This work has also been approved by the Ethics Committee on Animal Use from the University of Sa˜o Paulo (Protocol no. 11.1.308.53.9). All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs Diazepam (Roche, Brazil) was dissolved in a sterile saline solution and given i.p. 30 min prior the test at the doses of
Fig. 1. (A) The experimental box used for the Light Switch-off Test, displaying the light stimulus. (B) Switch-off responses to the light stimulus (SOR) of rats exposed to the standard protocol of the SOR test (n = 8) compared with a group only exposed to the dark chamber (n = 6). Rats actively cross compartments of the chamber in order to switch the light off. *Indicates statistical significant difference between groups across the four blocks of ten trials each (repeated measure two-way ANOVA).
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Experimental protocol Each test session consisted of 40 light stimuli delivered between random intervals (ranging from 15 to 45 s). Rats were placed individually into the experimental chamber and acclimatized for 5 min before the test started. Once the light stimulus was delivered, the rat was able to turn it off by crossing from one compartment to the other. This escape reaction was recorded as a ‘‘‘‘SOR” since it occurred within the 20-s length of the light stimulus. The number of crossings during the absence of light stimulus was registered as a locomotion index. Thus, each session consisted of 40 successive trials, separated by an interval in which the light remained switched off (dark trial) so that each session lasted for about 40 min. The number of shuttling responses during the light and dark components of the test was collected by software, which controlled the presentation and termination of the stimuli. The presentation and sequencing of the light stimuli were also controlled by the same software, which also collected data in blocks of 10 trials (blocks 1, 2, 3 and 4) during the entire session. Each animal was submitted to only one session and the chamber was cleaned with 20% ethanol after each session.
between groups (F1,36 = 8.3, p = 0.01) and blocks (F3,36 = 10.2, p < 0.001). There is no significant interaction between groups and blocks. The pro-aversive drug mCPP at a dose of 1 mg/kg caused a marked increase in the total number of SOR (F4,35 = 3.15; p = 0.02, Fig. 2A). This effect cannot be attributed to a general boost of motor activity, since the locomotion index did not differ between groups (F4,35 = 1.12, p = 0.36, Fig. 2B). Regarding the latency
Statistical analysis Data obtained from the independent groups of animals tested in the LSOT were submitted to a two-way repeated measure analysis of variance (ANOVA) followed by Bonferroni post hoc test, considering groups (dark and light) and blocks of ten trials (B1, B2, B3, B4) as the factors. The same analysis was performed on the data obtained (SORs, intertrial activity and latency to respond to the light) with drug treatments. Frequencies of avoidance responses across the four blocks were subjected to a repeated measure two-way ANOVA using the treatment as the between factor and blocks of 10 trials each as the within and repeated measure factor. One-way ANOVA was also performed on the total number of SORs, intertrial activity and latency to switch-off the light recorded for the treated groups of rats. All values are reported as mean ± standard error of mean (SEM). A level of p < 0.05 was used to confirm statistically significant differences.
RESULTS A starting point to the investigation was to find out if light by itself was a stimulus aversive enough to trigger a defensive response. We tested an additional control group of animals (n = 6) in the same protocol except for the absence of any light stimulation. Thus, this group’s total frequency of SOR would be the sum of the random crossings from exploratory behavior during the whole duration of the test. Rats of the light group crossed the divisory line of the chamber more than this control group. Thus, SOR is not an aleatory phenomenon; rats actively cross compartments of the chamber in order to switch the light off (Fig. 1B). Two-way repeated measures ANOVA indicated significant differences
Fig. 2. (A) Total frequency of switch-off responses (SOR) for all groups of rats (n = 8 per group). Diazepam (Dz) did not attenuate this response while the serotonergic agonist mCPP at the dose of 1 mg/kg showed pro-aversive effects, increasing the frequency of SORs. (B) Total number of intertrial crossings, an index of locomotor activity. Diazepam and mCPP did not change the motor activity of the animals during the intertrial period. (C) Mean latency of switch-off responses to the light. A similar latency pattern was found across all groups. Once the response is elicited, it will happen up to the 10th second of the stimulus presentation. *Significant different from the control V (vehicle). #Significant difference from diazepam.
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to respond, a uniform time window was found across all groups. The animal’s response to cease the light occurred within the first 10 seconds of light stimulation (F4,35 = 0.92; p = 0.46; Fig. 2C). Depending on the frequency of SOR, each test may last for about 35–40 min. Thus, we divided each session in four blocks of 10 stimuli each, to observe how the studied parameters are displayed throughout the duration of the session. Significant effects on SORs were caused by treatments (F4,105 = 3.15, p < 0.05), blocks (F3,105 = 9.16, p < 0.01) and interaction between treatments and blocks (F12,105 = 2.00 p < 0.05). Fig. 3A shows how the SOR is maintained along the time course of the test in the control group while it was clearly increased in the group of rats under mCPP at 2.0 mg/kg. Interestingly, the novelty-induced exploration during the first block was significantly decreased in the diazepam 2 mg/kg and mCPP 1 mg/kg groups, but this locomotor index remained stable for the remaining blocks. There was significant effects on blocks (F3,105 = 16.22, p < 0.05) as well as on interaction between blocks and treatments (F12,105 = 2.21, p < 0.05) but there was no significant effect of treatments on the intertrial activity (F4,105 = 1.12, p > 0.05) (Fig. 3B). No significant effect of treatments (F4,35 = 0.21, p > 0.05), blocks (F3,105 = 1.79, p > 0.05) nor interaction between treatments and blocks (F12,105 = 0.73, p > 0.05) were found on the latencies to switch off the light. Also, none of the drugs (at doses used in this study) altered the latency to switch off the light across blocks in all groups (Fig. 3C).
DISCUSSION The LSOLT was first designed and used in 2004 when it yielded interesting results on the participation of dopaminergic mechanisms in the SOR expression (Reis et al., 2004). Unlike the light/dark test which is based on the ethological view of shifting relative propensities to explore and to retreat from an unknown space (Crawley and Goodwin, 1980), the LSOT uses an unconditioned luminous stimulus aversive enough to trigger an escape response of the rat. Recently, our group has been conducting a series of tests to achieve the optimum protocol for reliable and replicable results. One of the important variables is the presence of the acrylic portal that clearly indicates to the rodent the existence of two compartments. The absence of this division has been shown to alter the SOR. So far, the aforementioned protocol described here has proven effective in other studies conducted by our group. Both systemic injections and local microinjection (into the dorsolateral superior colliculus) of the selective dopamine D2 receptor antagonist sulpiride increased the number of SORs (Reis et al., 2004; Muthuraju et al., 2016). We also addressed the hypothesis that the crossings were a mere product of casualty, by testing animals in the same experimental box and with the same software record system, but with disconnected bulbs so no light
Fig. 3. (A) Switch-off responses for all groups, clustered into four blocks (1, 2, 3 and 4) of 10 stimuli each per session. There was significant effect of blocks, treatments and interaction between blocks and treatments. The group treated with mCPP 1 mg/kg produced high frequency of responses on the third and fourth blocks compared to the V (vehicle) group. (B) Intertrial crossings as observed in each block. The novelty-induced exploration during the first block was significantly reduced in the diazepam 2 mg/kg and mCPP 1 mg/kg groups, but this locomotor index remained stable for the remaining blocks. (C) Latency to switch-off the light. No significant difference was found between groups regarding the latency to respond to the light stimulus. Note that the SOR latencies remain relatively stable across the blocks in all groups tested. Dz: diazepam. *Significant difference from the control group in the same block.
would be presented during the session. According to our results, the aversiveness of luminous stimulus is discriminated by the animal, which actively displays a response in order to cease it and this response is not delivered by chance. We are currently working on an increasing scale of light intensities that could reveal the amount of lux necessary to the luminous stimulus to be considered aversive by the rat.
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As this test is dependent on motor behavior, another important aspect to be clarified was if there were any locomotor changes that could interfere with the SOR. None of the drugs at the doses tested were capable of altering the total locomotor index, which strengthens the relevance of the SOR as a defensive behavior. An interesting finding was obtained from the block analysis for locomotion in the groups diazepam 2 mg/kg and mCPP 1 mg/kg. Apparently, these drugs curbed the novelty induced locomotor peak in the first block of the test, but did not alter the total count. However, due to its sedative property, we believe that higher doses of diazepam would impair SOR. To conclude, mCPP at the dose of 1 mg/kg was effective in increasing and keeping the high frequency of SORs, especially in the third and fourth blocks, when other groups tend to decrease the response. Compared to other uses of mCPP as an inductor of obsessive–compulsive behavior, this effective dose was relatively low (Papakosta et al., 2013; Tucci et al., 2015). In the present study, the switch off responses were not affected by the benzodiazepine agent diazepam given at doses (1 and 2 mg/kg) known to be highly effective in animal models of generalized anxiety such as elevated plus-maze, Geller-Seifter conflict test, dark-light test and others (Howard et al., 1982; Pellow et al., 1985; Cole and Rodgers, 1995; Chaouloff et al., 1997). This finding is coherent with several reports showing that the administration of benzodiazepines does not reduce unconditioned fear responses, as it is the case with the LSOT. On the other hand, there is general agreement on the idea that overall stimulation of the 5-HT system by serotoninergic agonists, such as mCPP, yields an anxiogenic profile (Kennett et al., 1989; Lin and Parsons, 2002; Birkett et al., 2011). The results boost our interest in the ongoing evaluation of light aversion. In the LSOT the escape from illuminated areas is considered to be an innate response with an evolutionary basis; that is, rodents are nocturnal and are more vulnerable in the light (Crawley and Goodwin, 1980; Bourin, 2015; Bourin and Hascoet, 2003; Reis et al., 2004). However, it remains to be assessed how aversive are such light exposure trials as compared to other aversive stimuli. Would it be possible that the magnitude of the aversiveness triggered by light would also be dependent on the conditions in which it is assessed (i.e. amount of luminance delivered by the light bulb, number of stimuli, housing conditions in the vivarium where animals are kept etc.)? Further studies will be conducted to explore the potential of this test as an index of innate fear and sensorimotor activity. Thus, the LSOT can be used to measure rodent’s aversion to light and their defensive response to it. It constitutes a promising methodology to be further studied and systematized. Among its many advantages, the LSOT is a simple procedure, easily replicable, non-invasive and minimally stressful test, since it does not include foot shocks or excessively aversive conditions to be endured by the animals (Beauchamp and Morton, 2015). The activation of the behavioral inhibition system (septo-hippocampal system) leads to behavioral
inhibition, increment of arousal and increased attention (McNaughton and Gray, 2000). Anti-anxiety drugs like the benzodiazepines impair the activity of this system by increasing rodent´s motor activity. In the present case, the benzodiazepine compound midazolam did not interfere with the motor activity of the animals submitted to the LSOT. This lack of effect supports the notion that there is more than one state of fear organized in other neural substrates. The dorsal periaqueductal gray and the superior colliculus are strong candidates for being part of this system since dopaminergic drugs locally injected into these structures change the light SORs emitted by rodents in our experimental conditions (Muthuraju et al., 2016). It is important to note that, in contrast to the resistance to the ‘‘anxiolytic-like” effects of benzodiazepines the present test showed a clear response to the serotoninergic agent mCPP. Taking into account that although some classical animal models of anxiety, such as the elevated plus-maze, show good sensitivity to the anxiolytic action of benzodiazepines they are not sensitive to other classes of drugs, which proved to be ‘‘anxiolytic” in the clinics, such as serotonergic compounds. Thus, it is fortunate to have at our disposal a simple and reliable test that measures innate fear with good sensitivity to serotoninergic drugs, which has increasingly been used in panic disorders, phobias and other types of anxiety disorders.
CONCLUSION Nowadays it is known that truly novel, clinically drug targets intended to treat psychiatric disorders have not been successfully translated from the neuropsychopharmacology laboratory to the clinic. As a consequence, this has dampened some pharmaceutical companies to move forward with new projects for psychiatric disorders. Therefore, given that the current preclinical models of anxiety have not been successful in predicting effects in humans all efforts to bring a new animal model of anxiety state based on unconditioned fear are welcome.
CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest.
FUNDING This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil. V. M. Saito was supported by FAPESP Fellowship (Process 2012/03707-5). Acknowledgments—VMS performed the experiments and wrote the first draft of the manuscript; MLB designed the test protocol, supervised the experiments and wrote/proof read the manuscript.
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(Accepted 26 July 2016) (Available online 2 August 2016)
THE LIGHT SWITCH-OFF RESPONSE AS A PUTATIVE RODENT TEST OF INNATE FEAR VIVIANE M. SAITO * AND MARCUS L. BRANDA˜O
also being considered a controversial issue. The use of experimental animals has been based on factual and moral assumptions, in which it is thought that animal models are reliable to predict results that will be obtained in human studies and that are morally justifiable to avoid atrocities in human subjects, as it happened in the II World War (Ferdowsian and Gluck, 2015). One of the drawbacks pointed is that behavioral tests often present limited explanatory power, since they measure behavior as an indicator, in an attempt to approach the functional role of the phenomena observed. Recent worldwide discussion on the ethical issues in animal experimentation (Nordgren, 2002; Akhtar, 2015; Ferdowsian and Gluck, 2015) instigates the refinement for methods used in neurobiology of behaviors as fear and anxiety. The concept of fear and anxiety is in itself a matter of debate (LeDoux, 2014; Perusini and Fanselow, 2015), and more so when both concepts are transposed to animal behavior. One of the most reliable tests used to predict the anxiolytic- and anxiogenic-like effects of drugs in rodents is the light/dark test, a proposed ‘‘exploratory” animal behavior model for the anxiolytic action of benzodiazepines (Crawley and Goodwin, 1980). The main parameters to assess the anxiolytic profile of drug treatment are the number of transitions between the two compartments, the latency time for the first passage from the light compartment to the dark one, the movement in each compartment, and the time spent in each compartment. Transitions in this test are considered an index of activity/exploration whereas the time spent in each compartment reflects aversion/attraction (Pitsikas et al., 2008; Bourin, 2015). Although this test exploits the differences in behavior seen in dark and lit areas, its rationale remains on the natural conflict between exploration and avoidance that arises when rodents are exposed to novel environments (Crawley and Goodwin, 1980). Our group has developed an animal test focused on the unconditional fear/anxiety paradigm, continuing the first study by Reis et al. (2004). The Light Switch-Off Test (LSOT) is based on the innate motivation to cease an aversive stimulus (bright light). Light by itself is a complex stimulus with both luminance and thermal components (Burns and Webb, 1994). In our protocol, the luminous stimulus per se evokes the light switch-off response (SOR), which is expressed when the rat crosses from one side to the other in a shuttle box. There is no need for previous conditioning or foot shocks. Since the animal emits a response to reduce its exposure to that stimulus,
Laboratory of Neuropsychopharmacology, FFCLRP, Sa˜o Paulo University, Campus USP, Ribeira˜o Preto, Sa˜o Paulo 14040-901, Brazil Instituto de Neurocieˆncias e Comportamento, Avenida do Cafe´, 2450, Ribeira˜o Preto, SP 14050-000, Brazil
Abstract—Recent discussions on the ethics in animal experimentation instigate the refinement of methods used in Behavioral Neuroscience, particularly regarding fear/anxiety paradigms. We propose the Light Switch-Off Test (LSOT), based on the innate motivation to cease an aversive stimulus (bright light), displayed naturally by rodents in their habitat. Forty-six male adult Wistar rats were allocated into independent groups: control, diazepam at 1 or 2 mg/kg, and meta-Chlorophenylpiperazine (mCPP) at 0.5 or 1 mg/kg. The experimental box has two square compartments, separated by an acrylic portal. In each side of the box, there is a 40-W incandescent light bulb. After a habituation period in the box, 40 light stimuli (trials lasting up to 20 s each) are emitted at random intervals. By crossing compartments during the lighted period, the rat could switch-off the stimulus. Parameters observed are the number of switch-off responses (SORs), latency of SOR and intertrial locomotion. The SOR frequency was higher in rats treated with mCPP at 1 mg/kg, an anxiogenic drug, while diazepam at the doses used in this study did not produce effects. Animals exposed solely to the box for the length of the test did not respond in a false positive way. Therefore, the SOR represents a good index to measure the innate rodent fear of bright-lighten areas, once they react quickly in order to turn off the stimulus. Among its many advantages, the LSOT is a simple, replicable, non-invasive and minimally stressful procedure, since it does not expose animals to excessively aversive stimulus. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: animal test, behavior, fear, rat, diazepam, mCPP.
INTRODUCTION Animal models are widely used to model clinical conditions for basic research (Sousa et al., 2006) though *Correspondence to: V. M. Saito, Laboratory of Neuropsychopharmacology, FFCLRP, Sa˜o Paulo University, Campus USP, Ribeira˜o Preto, Sa˜o Paulo 14040-901, Brazil. Fax: +55-16-33154830. E-mail address: [email protected] (V. M. Saito). Abbreviations: 5-HT, serotonin; ANOVA, analysis of variance; LSOT, Light Switch-Off Test; mCPP, meta-Chlorophenylpiperazine; SOR, switch-off response. http://dx.doi.org/10.1016/j.neuroscience.2016.07.044 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 160
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the bright light used in this protocol apparently served as an aversive stimulus. The response to light is naturally occurring and belongs to the ethological repertoire of rodents in their habitat. In the experimental setup, lever pressing was readily acquired and maintained when each press turned off a lamp for 1 min (Keller, 1941). Other study showed that the same learned response ranged in an inverted U-shaped function of light intensity, with intermediate intensities eliciting the highest response rates (Kaplan et al., 1965). The importance of vision is highlighted in the aversion of open spaces that rodents display when submitted to the elevated plus maze, a traditional unconditioned model for anxiety- and conflict-like behaviors (Garcia et al., 2005). In settings where auditory and olfactory stimuli are controlled, aversion is triggered by the light penetration and image formation in the retina (Morato, 2006). One of the arguments that support the intense light as an aversive stimulus to albino rats is the lack of pigment in their iris and choroid, which reduces their vision adaptation and predisposes to visual damage (Stryjek et al., 2013). Although there is a body of evidence indicating that light is an aversive stimulus by itself (Stern and Laties, 1989, 1998; Garcia et al., 2005), no standardized test protocol has been developed using only light as aversive stimulus to trigger an innate response from the rat. Usually, light is used as conditioned cue that precedes the aversive/rewarding stimulus in other protocols (Cassaday and Thur, 2015; Pezze et al., 2016). Here, we present the protocol that has been used and an initial screening of the effects of two drugs in the test: diazepam (a well-known benzodiazepine used as anxiolytic in clinical practice) and meta-Chlorophenylpiperazine (mCPP), a serotoninergic drug with anxiogenic properties, pharmacologically classified as a non-specific agonist for serotonin (5-HT) receptors (Kennett et al., 1989; Kahn and Wetzler, 1991).
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1 or 2 mg/kg. These doses were used based on previous studies in the elevated plus maze that did not induce sedation and motor impairment. mCPP (RBI, MA, USA) was also dissolved in physiological saline solution and delivered i.p. 15 min before the test, at the doses of 0.5 or 1 mg/kg, based on the minimal dose needed for proaversive effects obtained by Reimer et al. (2015, unpublished data). The control group received i.p. treatment with physiological saline. A group of animals tested in the chamber without any stimulus (‘‘Dark only” group) did not receive any treatment. Apparatus The experimental chamber consisted of a shuttle box comprising two compartments measuring 30 25 25 cm (Insight, Brazil). The side and back walls of the chamber were constructed of black Plexiglas and the ceiling and front door were made of transparent Plexiglas. The chamber was divided by an opaque acrylic portal to allow free exploration. A grid floor comprised 15 stainless steel rods with 2.0 mm diameter, spaced 1.2 mm apart. Two 40 W light bulbs were centered on each side of the rear of the chamber, 12 cm from the floor (Fig. 1A). The light was turned on and off noiselessly. The software and an appropriate interface connected to a PC provided by the manufacturer of the equipment (Esquiva Ativa; Insight, Brazil) allowed for recording and analysis of the frequencies and latency of escape responses as well as the intertrial locomotor activity.
EXPERIMENTAL PROCEDURES Animals A total of 46 male adult Wistar rats (weighting between 260 and 280 g) were used in this study. Animals were housed in collective cages in the colony room (12 h light–dark cycle in a temperature controlled, ventilated room). The experiments were carried out during the light phase of the cycle, between 08:00 a.m. and 17:00 p.m. All procedures followed the guidelines on the ethical use of animals by the Brazilian Society of Neuroscience and Behavior (SBNeC) which follows the National Institutes of Health (NIH) guide for the care and use of laboratory animals. This work has also been approved by the Ethics Committee on Animal Use from the University of Sa˜o Paulo (Protocol no. 11.1.308.53.9). All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs Diazepam (Roche, Brazil) was dissolved in a sterile saline solution and given i.p. 30 min prior the test at the doses of
Fig. 1. (A) The experimental box used for the Light Switch-off Test, displaying the light stimulus. (B) Switch-off responses to the light stimulus (SOR) of rats exposed to the standard protocol of the SOR test (n = 8) compared with a group only exposed to the dark chamber (n = 6). Rats actively cross compartments of the chamber in order to switch the light off. *Indicates statistical significant difference between groups across the four blocks of ten trials each (repeated measure two-way ANOVA).
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Experimental protocol Each test session consisted of 40 light stimuli delivered between random intervals (ranging from 15 to 45 s). Rats were placed individually into the experimental chamber and acclimatized for 5 min before the test started. Once the light stimulus was delivered, the rat was able to turn it off by crossing from one compartment to the other. This escape reaction was recorded as a ‘‘‘‘SOR” since it occurred within the 20-s length of the light stimulus. The number of crossings during the absence of light stimulus was registered as a locomotion index. Thus, each session consisted of 40 successive trials, separated by an interval in which the light remained switched off (dark trial) so that each session lasted for about 40 min. The number of shuttling responses during the light and dark components of the test was collected by software, which controlled the presentation and termination of the stimuli. The presentation and sequencing of the light stimuli were also controlled by the same software, which also collected data in blocks of 10 trials (blocks 1, 2, 3 and 4) during the entire session. Each animal was submitted to only one session and the chamber was cleaned with 20% ethanol after each session.
between groups (F1,36 = 8.3, p = 0.01) and blocks (F3,36 = 10.2, p < 0.001). There is no significant interaction between groups and blocks. The pro-aversive drug mCPP at a dose of 1 mg/kg caused a marked increase in the total number of SOR (F4,35 = 3.15; p = 0.02, Fig. 2A). This effect cannot be attributed to a general boost of motor activity, since the locomotion index did not differ between groups (F4,35 = 1.12, p = 0.36, Fig. 2B). Regarding the latency
Statistical analysis Data obtained from the independent groups of animals tested in the LSOT were submitted to a two-way repeated measure analysis of variance (ANOVA) followed by Bonferroni post hoc test, considering groups (dark and light) and blocks of ten trials (B1, B2, B3, B4) as the factors. The same analysis was performed on the data obtained (SORs, intertrial activity and latency to respond to the light) with drug treatments. Frequencies of avoidance responses across the four blocks were subjected to a repeated measure two-way ANOVA using the treatment as the between factor and blocks of 10 trials each as the within and repeated measure factor. One-way ANOVA was also performed on the total number of SORs, intertrial activity and latency to switch-off the light recorded for the treated groups of rats. All values are reported as mean ± standard error of mean (SEM). A level of p < 0.05 was used to confirm statistically significant differences.
RESULTS A starting point to the investigation was to find out if light by itself was a stimulus aversive enough to trigger a defensive response. We tested an additional control group of animals (n = 6) in the same protocol except for the absence of any light stimulation. Thus, this group’s total frequency of SOR would be the sum of the random crossings from exploratory behavior during the whole duration of the test. Rats of the light group crossed the divisory line of the chamber more than this control group. Thus, SOR is not an aleatory phenomenon; rats actively cross compartments of the chamber in order to switch the light off (Fig. 1B). Two-way repeated measures ANOVA indicated significant differences
Fig. 2. (A) Total frequency of switch-off responses (SOR) for all groups of rats (n = 8 per group). Diazepam (Dz) did not attenuate this response while the serotonergic agonist mCPP at the dose of 1 mg/kg showed pro-aversive effects, increasing the frequency of SORs. (B) Total number of intertrial crossings, an index of locomotor activity. Diazepam and mCPP did not change the motor activity of the animals during the intertrial period. (C) Mean latency of switch-off responses to the light. A similar latency pattern was found across all groups. Once the response is elicited, it will happen up to the 10th second of the stimulus presentation. *Significant different from the control V (vehicle). #Significant difference from diazepam.
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to respond, a uniform time window was found across all groups. The animal’s response to cease the light occurred within the first 10 seconds of light stimulation (F4,35 = 0.92; p = 0.46; Fig. 2C). Depending on the frequency of SOR, each test may last for about 35–40 min. Thus, we divided each session in four blocks of 10 stimuli each, to observe how the studied parameters are displayed throughout the duration of the session. Significant effects on SORs were caused by treatments (F4,105 = 3.15, p < 0.05), blocks (F3,105 = 9.16, p < 0.01) and interaction between treatments and blocks (F12,105 = 2.00 p < 0.05). Fig. 3A shows how the SOR is maintained along the time course of the test in the control group while it was clearly increased in the group of rats under mCPP at 2.0 mg/kg. Interestingly, the novelty-induced exploration during the first block was significantly decreased in the diazepam 2 mg/kg and mCPP 1 mg/kg groups, but this locomotor index remained stable for the remaining blocks. There was significant effects on blocks (F3,105 = 16.22, p < 0.05) as well as on interaction between blocks and treatments (F12,105 = 2.21, p < 0.05) but there was no significant effect of treatments on the intertrial activity (F4,105 = 1.12, p > 0.05) (Fig. 3B). No significant effect of treatments (F4,35 = 0.21, p > 0.05), blocks (F3,105 = 1.79, p > 0.05) nor interaction between treatments and blocks (F12,105 = 0.73, p > 0.05) were found on the latencies to switch off the light. Also, none of the drugs (at doses used in this study) altered the latency to switch off the light across blocks in all groups (Fig. 3C).
DISCUSSION The LSOLT was first designed and used in 2004 when it yielded interesting results on the participation of dopaminergic mechanisms in the SOR expression (Reis et al., 2004). Unlike the light/dark test which is based on the ethological view of shifting relative propensities to explore and to retreat from an unknown space (Crawley and Goodwin, 1980), the LSOT uses an unconditioned luminous stimulus aversive enough to trigger an escape response of the rat. Recently, our group has been conducting a series of tests to achieve the optimum protocol for reliable and replicable results. One of the important variables is the presence of the acrylic portal that clearly indicates to the rodent the existence of two compartments. The absence of this division has been shown to alter the SOR. So far, the aforementioned protocol described here has proven effective in other studies conducted by our group. Both systemic injections and local microinjection (into the dorsolateral superior colliculus) of the selective dopamine D2 receptor antagonist sulpiride increased the number of SORs (Reis et al., 2004; Muthuraju et al., 2016). We also addressed the hypothesis that the crossings were a mere product of casualty, by testing animals in the same experimental box and with the same software record system, but with disconnected bulbs so no light
Fig. 3. (A) Switch-off responses for all groups, clustered into four blocks (1, 2, 3 and 4) of 10 stimuli each per session. There was significant effect of blocks, treatments and interaction between blocks and treatments. The group treated with mCPP 1 mg/kg produced high frequency of responses on the third and fourth blocks compared to the V (vehicle) group. (B) Intertrial crossings as observed in each block. The novelty-induced exploration during the first block was significantly reduced in the diazepam 2 mg/kg and mCPP 1 mg/kg groups, but this locomotor index remained stable for the remaining blocks. (C) Latency to switch-off the light. No significant difference was found between groups regarding the latency to respond to the light stimulus. Note that the SOR latencies remain relatively stable across the blocks in all groups tested. Dz: diazepam. *Significant difference from the control group in the same block.
would be presented during the session. According to our results, the aversiveness of luminous stimulus is discriminated by the animal, which actively displays a response in order to cease it and this response is not delivered by chance. We are currently working on an increasing scale of light intensities that could reveal the amount of lux necessary to the luminous stimulus to be considered aversive by the rat.
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As this test is dependent on motor behavior, another important aspect to be clarified was if there were any locomotor changes that could interfere with the SOR. None of the drugs at the doses tested were capable of altering the total locomotor index, which strengthens the relevance of the SOR as a defensive behavior. An interesting finding was obtained from the block analysis for locomotion in the groups diazepam 2 mg/kg and mCPP 1 mg/kg. Apparently, these drugs curbed the novelty induced locomotor peak in the first block of the test, but did not alter the total count. However, due to its sedative property, we believe that higher doses of diazepam would impair SOR. To conclude, mCPP at the dose of 1 mg/kg was effective in increasing and keeping the high frequency of SORs, especially in the third and fourth blocks, when other groups tend to decrease the response. Compared to other uses of mCPP as an inductor of obsessive–compulsive behavior, this effective dose was relatively low (Papakosta et al., 2013; Tucci et al., 2015). In the present study, the switch off responses were not affected by the benzodiazepine agent diazepam given at doses (1 and 2 mg/kg) known to be highly effective in animal models of generalized anxiety such as elevated plus-maze, Geller-Seifter conflict test, dark-light test and others (Howard et al., 1982; Pellow et al., 1985; Cole and Rodgers, 1995; Chaouloff et al., 1997). This finding is coherent with several reports showing that the administration of benzodiazepines does not reduce unconditioned fear responses, as it is the case with the LSOT. On the other hand, there is general agreement on the idea that overall stimulation of the 5-HT system by serotoninergic agonists, such as mCPP, yields an anxiogenic profile (Kennett et al., 1989; Lin and Parsons, 2002; Birkett et al., 2011). The results boost our interest in the ongoing evaluation of light aversion. In the LSOT the escape from illuminated areas is considered to be an innate response with an evolutionary basis; that is, rodents are nocturnal and are more vulnerable in the light (Crawley and Goodwin, 1980; Bourin, 2015; Bourin and Hascoet, 2003; Reis et al., 2004). However, it remains to be assessed how aversive are such light exposure trials as compared to other aversive stimuli. Would it be possible that the magnitude of the aversiveness triggered by light would also be dependent on the conditions in which it is assessed (i.e. amount of luminance delivered by the light bulb, number of stimuli, housing conditions in the vivarium where animals are kept etc.)? Further studies will be conducted to explore the potential of this test as an index of innate fear and sensorimotor activity. Thus, the LSOT can be used to measure rodent’s aversion to light and their defensive response to it. It constitutes a promising methodology to be further studied and systematized. Among its many advantages, the LSOT is a simple procedure, easily replicable, non-invasive and minimally stressful test, since it does not include foot shocks or excessively aversive conditions to be endured by the animals (Beauchamp and Morton, 2015). The activation of the behavioral inhibition system (septo-hippocampal system) leads to behavioral
inhibition, increment of arousal and increased attention (McNaughton and Gray, 2000). Anti-anxiety drugs like the benzodiazepines impair the activity of this system by increasing rodent´s motor activity. In the present case, the benzodiazepine compound midazolam did not interfere with the motor activity of the animals submitted to the LSOT. This lack of effect supports the notion that there is more than one state of fear organized in other neural substrates. The dorsal periaqueductal gray and the superior colliculus are strong candidates for being part of this system since dopaminergic drugs locally injected into these structures change the light SORs emitted by rodents in our experimental conditions (Muthuraju et al., 2016). It is important to note that, in contrast to the resistance to the ‘‘anxiolytic-like” effects of benzodiazepines the present test showed a clear response to the serotoninergic agent mCPP. Taking into account that although some classical animal models of anxiety, such as the elevated plus-maze, show good sensitivity to the anxiolytic action of benzodiazepines they are not sensitive to other classes of drugs, which proved to be ‘‘anxiolytic” in the clinics, such as serotonergic compounds. Thus, it is fortunate to have at our disposal a simple and reliable test that measures innate fear with good sensitivity to serotoninergic drugs, which has increasingly been used in panic disorders, phobias and other types of anxiety disorders.
CONCLUSION Nowadays it is known that truly novel, clinically drug targets intended to treat psychiatric disorders have not been successfully translated from the neuropsychopharmacology laboratory to the clinic. As a consequence, this has dampened some pharmaceutical companies to move forward with new projects for psychiatric disorders. Therefore, given that the current preclinical models of anxiety have not been successful in predicting effects in humans all efforts to bring a new animal model of anxiety state based on unconditioned fear are welcome.
CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest.
FUNDING This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil. V. M. Saito was supported by FAPESP Fellowship (Process 2012/03707-5). Acknowledgments—VMS performed the experiments and wrote the first draft of the manuscript; MLB designed the test protocol, supervised the experiments and wrote/proof read the manuscript.
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(Accepted 26 July 2016) (Available online 2 August 2016)