PNP-08581; No of Pages 7 Progress in Neuro-Psychopharmacology & Biological Psychiatry xxx (2014) xxx–xxx
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A new model to study visual attention in zebrafish Daniela Braida a,b, Luisa Ponzoni a,c, Roberta Martucci a, Mariaelvina Sala a,d,⁎ a
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy Fondazione IRCCS Don Gnocchi, Milan, Italy c Fondazione Fratelli Confalonieri, Milan, Italy d CNR, Institute of Neuroscience, Milan, Italy b
a r t i c l e
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Article history: Received 4 February 2014 Received in revised form 14 March 2014 Accepted 14 March 2014 Available online xxxx Keywords: Cholinergic system Learning and memory Moving shapes Teleost Visual attention
a b s t r a c t The major part of cognitive tasks applied to zebrafish has not fully assessed their attentional ability, a process by which the nervous system learns, organizes sensory input and generates coordinated behaviour. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply a modified version of novel object recognition test named virtual object recognition test (VORT) using 2D geometrical shapes (square, triangle, circle, cross, etc.) on two iPod 3.5-inch widescreen displays, located on two opposite walls of the water tank. Each fish was subjected to a familiarization trial (T1), and after different time delays (from 5 min to 96 h) to a novel shape recognition trial (T2). A progressive decrease, across time, of memory performance, in terms of mean discrimination index and mean exploration time, was shown. The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index. Zebrafish discriminated different movements (vertical, horizontal, oblique) and the discrimination index increased significantly when moving poorly discriminated shapes were presented, thus increasing visual attention. Taken together these findings demonstrate that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and for predicting the efficacy of pharmacotherapies for cognitive disorders. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Zebrafish are providing an attractive model for behavioural studies. Although these fish are relative newcomers to studies of learning and memory (Sison et al., 2006), a number of studies revealed that they are capable of performing well in a range of learning tasks such as avoidance learning (Blank et al., 2009; Morin et al., 2013), olfactory conditioning (Braubach et al., 2009), shuttle box alternation learning (Pather and Gerlai, 2009), place conditioning (Eddins et al., 2009), appetitive choice discrimination (Bilotta et al., 2005), visual discrimination learning (Colwill et al., 2005), associative conditioning task (Luchiari and Chacon, 2013), active avoidance conditioning (Xu et al., 2007), alternation based spatial memory task (Williams et al., 2002) and even automated learning paradigm (Hicks et al., 2006). Sison and Gerlai (2011) have designed an associative learning task that was deliberately made to resemble a classical radial arm maze in which the traditional cue (food reward) was replaced by the sight of conspecifics. Parker Abbreviations: VORT, Virtual Object Recognition Test; 2D, bidimensional; T1, Familiarization trial; T2, Recognition Trial; IP, Intraperitoneally; 3D, Tridimensional; NOR, Novel Object Recognition Test; WT, Wild Type; NIC, Nicotine; SCOP, Scopolamine; MEC, Mecamylamine. ⁎ Corresponding author at: Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy. Tel.: + 39 02 50317042; fax: +39 02 50317036. E-mail address: [email protected] (M. Sala).
et al. (2012) have developed a 3-choice serial reaction time task (3CSRTT) in which zebrafish could learn to perform a complex operant task, which requires more than 20 days, similar to that developed for rodents to test sustained attention and impulsivity. More recently, a new T-maze task has been set up (Braida et al., 2013a). Zebrafish were trained to reach a reservoir and stay for at least 20 s. The running time difference between the first and the second trial was calculated as a measure of memory of the spatial location of reward. In these conditions nicotine enhanced memory while scopolamine impaired it. As recently reported by Echevarria et al. (2011), apart from the 3CSRTT, the majority of the above mentioned tasks have not fully assessed attentional ability, a process by which the organism learns and the nervous system receives or acquires sensory input and generates coordinated behaviour of animals (Bushnell, 1998). Cognitive impairment is a core symptom of neuropsychiatric and neurologic disorders. Attentional deficit has been considered to be one of the most consistent areas of impairment associated with psychiatric and neurodegenerative diseases (Kelip et al., 2008; Perry et al., 2000) as Parkinson's disease, Alzheimer, attention deficit hyperactivity, stroke, epilepsy (Biederman et al., 2006; Rubia et al., 2005; Sahakian et al., 1988; Stretton and Thompson, 2012) and schizophrenia (Lyon et al., 2012). Attention includes a series of cognitive processes and disorders related to attention may underlie cognitive dysfunctions. The novel object recognition (NOR) test evaluates an animal's attention that is elicited by the presentation of novel stimuli. Interest in NOR
http://dx.doi.org/10.1016/j.pnpbp.2014.03.010 0278-5846/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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is very recent and a large proportion of the literature on cognition has been dedicated to object and visual recognition in humans, pigeons and primates (Spetch et al., 2006; Wallis and Bülthoff, 1999). The principal advantage of the NOR test is the rapid testing sequence and no necessary training other than the initial exposure session. In a recent paper we created a modified version of NOR, named virtual object recognition test (VORT), in mice where the 3D objects were replaced with virtual stationary or moving geometrical 2D shapes presented on iPod screens (Braida et al., 2013b). This test, as that used in humans, is predominantly based on visual stimuli. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply VORT to fish either using stationary or moving 2D shapes. It is known that cortical acetylcholine release has been implicated in novelty-induced arousal, attention, the encoding of novel stimuli and memory consolidation (Acquas et al., 1998; Sarter and Bruno, 2000). Thus, to test the task validity, different cholinergic drugs were injected intraperitoneally (IP) to fish.
The shapes were looped on a 3rd generation iPod Touch (Apple) through iTunes for the duration of the experiment (320 pixels horizontal axis and 480 pixels vertical axis). The luminosity of the screens was constant across the two screens and testing sessions. After T1 the fish returned to the home tank. During T2 each fish was placed again in the central area after different time delays, from 5 min to 96 h, during which one of the two identical static familiar shapes was replaced with a novel one. The time delays and doses were chosen on the basis of our previous work, using the same task, in mice (Braida et al., 2013b). For dynamic cue the same or different shapes presented in T 1 moved in a different direction than in T1. Attention was paid to counterbalance the choice of the shapes. Within every time delay, all the pairing discriminated shapes were randomly tested. Shape recognition was manually scored with a stopwatch, by an experimenter blind to the treatment, in terms of exploration time whenever the zebrafish approached to the iPod area (10 cm) and directed its head toward the shape. All the fish were drug naive, and each fish was used only once. 9–11 fish per group were used.
2. Materials and methods
2.3. Drug and treatment
2.1. Animals
Body weight was measured as previously described (Braida et al., 2007). Fish were removed from their home tank using a net and placed in a container, filled with tank water, positioned on a digital balance. Zebrafish weight was determined as the weight of the container plus the fish minus the weight of the container before the fish was added. The mean of three measurements was recorded. All the drugs were injected IP in a volume of 2 μl/g using a Hamilton syringe, according to Braida et al. (2013a). All drugs were dissolved in sterile saline (0.9%) and were prepared fresh daily. The drugs were: nicotine bi-tartrate salt (Sigma–Aldrich, St.Louis, MO, USA), (0.02 mg/kg) given 20 min before the test; scopolamine hydrobromide (Sigma–Aldrich, St. Louis, MO, USA) (0.025 mg/kg) given 20 min before the test; and mecamylamine hydrochloride (Sigma–Aldrich, St. Louis, MO, USA) (0.1 mg/kg) given 30 min before the test. Control groups received sterile saline solution (0.9%) which was given 20 or 30 min before T1. The range of doses of nicotine and of antagonists were chosen on the basis of their activity on T-maze task and on the basis that they did not modify swimming behaviour (Braida et al., 2013a).
Adult short-finned wild-type (WT) zebrafish (Danio rerio) (0.4–1 g) of heterogeneous genetic background were obtained from a local aquarium supply store (Aquarium Center, Milan, Italy). Zebrafish were 6–12 months of age and were 3–4 cm long. In all experiments, the sex ratio of zebrafish was approximately 50–50%. Males and females were identified as previously reported (Braida et al., 2012). Fish were kept at approximately 28.5 °C on a 14:10-h light/dark cycle. Behavioural testing took place during the light phase between 09:00 and 14:00 h. Tank water consisted of deionized water and sea salts (0.6 g/10 L of water; Instant Ocean, Aquarium Systems, Sarrebourg, France). Approximately 30 adult fish were maintained in 96 L home tanks (75 cm long, 32 cm wide and 40 cm high) provided with constant filtration and aeration. Animals were acclimated for at least 2 weeks before the experiments. Fish were fed daily with brine shrimp and flake fish food (tropical fish food, Consorzio G5, Italy). The experimental protocol was approved by the Italian Governmental Decree No. 18/2013. All efforts were made to minimize the number of animals used and their discomfort.
2.4. Statistical analysis 2.2. Virtual object recognition test (VORT) A transparent Plexiglas tank (filled with tank water at a level of 10 cm) was used in VORT. The apparatus (Fig. A) was characterized by a rectangular environment (70 cm × 30 cm × 10 cm). After a week habituation to the apparatus, each fish was restricted for 5 min in a 20 cm central area delimited by two opaque barriers to visually isolate the stimuli areas where two identical white geometrical shapes on a black background were shown on two iPod 3.5-inch widescreen displays located externally to the opposite 10 cm wide walls. Then, each fish was subjected to a familiarization trial (T1), and after different time delays to a novel shape recognition trial (T2). Both T1 and T2 consisted of a 10 min session during which two identical white geometrical shapes on a black background were shown on two iPod screens. For static cue, shapes were simple geometrical shapes (square, triangle, circle, cross, etc.) with equal surface (2.5 cm2). A complete list is given in Fig. 2C. For dynamic cue, the same above shapes moved horizontally, vertically or diagonally (distance 320 px) at a constant speed of 120 px/s. The videos were created in Adobe Flash, with a frame size of 320 pixels × 480 pixels, with a rate of 30 frames per second. The videos were encoded with a video encoder H264 (Apple), a software program that converts information written in one format or code to a different one. Usually, this is carried out in order to standardize information, improve speed and security, and reduce the file in size which then saves space.
All data were expressed as mean ± S.E.M. Pair-wise comparisons were assessed with Student's t-test. Different groups were assessed by one-way analysis of variance (ANOVA) for multiple comparisons followed by Tukey's post-hoc test. Data were expressed as discrimination index [(time spent exploring novel shape − time exploring familiar shape) / (time spent exploring novel shape + time exploring familiar shape)], as previously described in mice (Braida et al., 2013b). Data from fish receiving saline 20 or 30 min before T1 were pooled after making sure that there was no statistical difference between the two groups. The level of significance was taken as p b 0.05. All statistical analyses were done using software Prism, version 6 (GraphPad, San Diego, CA, USA). 3. Results 3.1. Different time delays progressively decrease memory performance In Fig. 1 the effects of different time delays on VORT, in terms of discrimination index (Fig. 1A) and the exploration time respectively (Fig. 1B), are shown. One-way ANOVA showed a significant difference among groups in delay time (from 5 min to 96 h) (F(3,36) = 7.46; p b 0.0005). Post-hoc analysis revealed that the mean discrimination index, evaluated at 96 h, was significantly lower than that evaluated at the remaining intervals. Concerning the exploration time, one-way ANOVA revealed a significant difference among the groups (F(9,90) =
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 1. Performance evaluated in VORT (A–B) at increasing time delays. Mean discrimination index (A) and mean exploration time (B) evaluated when zebrafish were presented highly discriminated shapes. Data are expressed as mean ± S.E.M. N = 9–11 for each group. § p b 0.05, §§ p b 0.01 as compared to 96 h group; *p b 0.05, **p b 0.01, ***p b 0.001 as compared to corresponding familiar exploration time (Tukey's test).
6.21; p b 0.0001). Post-hoc analysis showed that the time spent to explore the new shape was higher than the familiar one at all the tested time delays except for 96 h. The best performance was obtained at the inter-trial of 5 min; thus, such delay was adopted in the following experiments. 3.2. Manipulation of cholinergic system alters the ability of fish to discriminate shapes On the basis of previous findings obtained in mice (Braida et al., 2013b) a number of shapes were tested for their ability to be discriminated by fish. Fig. 2C shows which shapes were easily discriminated (left) and which shapes were not (right). The obtained mean discrimination indices were significantly different (t = 5.47, p b 0.0001) (Fig. 2A). Accordingly, when using highly discriminated shapes more time was spent exploring the novel shape than the familiar one (F(3,36) = 3.53, p = 0.001) (Fig. 2B). In contrast, a similar mean exploration time for the familiar and the novel shapes, for the poorly discriminated shapes, was obtained (F(3,36) = 1.53, p = 0.81). The effects of different cholinergic treatments on VORT are shown in Fig. 3. We first investigated whether memory performance could be improved by nicotine treatment using shapes that were difficult to discriminate or whether further improved using highly discriminated shapes. Then we investigated whether treatment with scopolamine or mecamylamine impaired memory. Nicotine increased mean discrimination index compared to saline group (t = 3.55, p = 0.002). When fish were given different cholinergic antagonists there was a significant difference in the discrimination index among groups (F(3,36) = 18.17, p b 0.0001). Post-hoc tests revealed that, when discriminated shapes
Fig. 2. Performance evaluated in VORT using highly discriminated or poorly discriminated shapes. Mean discrimination index (A), mean exploration time (B) and examples of highly discriminated (left) and poorly discriminated (right) shapes used for the experiment (C). Data are expressed as mean ± S.E.M. N = 9–11 for each group. ****p b 0.0001 as compared to discriminated shapes (Student's t-test); @@@ p b 0.001 as compared to corresponding familiar shapes (T2) (Tukey's test).
were presented to zebrafish, both scopolamine and mecamylamine decreased mean discrimination index in comparison with saline, while treatment with nicotine did not further improve this parameter. One-way ANOVA revealed a significant difference among groups in the mean exploration time when poorly discriminated shapes were presented to the group of animals treated with nicotine (F(3,36) = 3.14, p = 0.003). Post-hoc tests revealed that, during the T2 phase, the mean exploration time of the novel shape was higher than the familiar one (Fig. 3C). When discriminated shapes were presented, one way ANOVA revealed a significant difference in the exploration time of zebrafish treated with saline (F(3,36) = 2.63, p = 0.0008) and nicotine (F(3,36) = 3.14, p = 0.003 (Fig. 3D). Tukey's test indicated that zebrafish spent more time exploring novel than familiar shape in these groups. 3.3. Zebrafish are able to discriminate different movements applied to shapes and the application of movement increases attention In Fig. 4 the effect of movement applied to discriminated shapes on VORT is reported. When a novel movement was applied during T2 to the same or different new shapes (Fig. 4C), a similar discrimination
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 3. Effect of different cholinergic drugs on mean (±S.E.M.) discrimination index (A and B) and exploration time (C and D) evaluated in VORT. Performance was assessed using poorly discriminated (left) and highly discriminated (right). Nicotine (NIC) (0.02 mg/kg) or scopolamine (SCOP) (0.025 mg/kg) was injected IP 20 min before T1; mecamylamine (MEC) (0.1 mg/kg) was injected IP 30 min before T1. Saline = pool of fish receiving saline 20 or 30 min before T1. N = 10 for each group. §§p b 0.01 as compared to corresponding saline group (Student's t-test); °°°p b 0.001, °°°°p b 0.0001 as compared to corresponding saline and nicotine groups; **p b 0.01 as compared to corresponding familiar shape (T2) (Tukey's test).
index was obtained (t = 0.96 n.s.), indicating that zebrafish were able to distinguish different kinds of movements (vertical, horizontal, oblique). ANOVA showed that the exploration time (Fig. 4B) was significantly higher in T2 than in T1 between the two conditions when a new movement was introduced (same shapes: F(3,68) = 3.14, p b 0.03; different shapes: F(3,128) = 3.62, p b 0.01). We investigated also the possibility that fish could improve their performance when the movements were applied to poorly discriminated stationary shapes. As reported in Fig. 5A, the mean discrimination index increased significantly when moving shapes were presented (F(2,27) = 63.6; p = 0.0001). Post-hoc comparison revealed that the application of movement significantly increased the mean discrimination index either if the movement was different from T1 or it was the same. No difference was found in the mean exploration time when stationary shapes were presented (F(3,36) = 1.54; p N 0.05) (Fig. 5B). In contrast, there was a difference among groups in the mean exploration time when both different (F(3,36) = 8.13; p b 0.0001) and the same (F(3,36) = 4.56; p b 0.001) movements were applied to shapes. Post-hoc test revealed that during T2 there was a significant increase in the mean exploration time of novel shapes compared to the familiar ones, independently of whether the movement was changed or not (in Fig. 5C). 4. Discussion Our results demonstrate, for the first time, the possibility to evaluate selective attention in zebrafish using a modified version of the well established NOR task, which was applied to rodents to assess recognition memory for 3D objects (Ennaceur and Delacour, 1988). Zebrafish, submitted to VORT, at different inter-trial time delays, showed a progressive memory decay which was maximal when the delay of 96 h was applied. The obtained discrimination indices were comparable to
those previously found in mice submitted to the same task (Braida et al., 2013b). Notably, the mean exploration time was maintained to a higher level (more than 200 s) in zebrafish than in mice (less than 50 s) at all the tested time intervals. These differences could be due to the higher exploration activity of fish, compared to rodents, in the aquatic environment or to a different visual acuity. Measurement of visual acuity and contrast sensitivity, based on the OptoMotry device (Tappeiner et al., 2012), revealed a remarkable higher visual ability in adult zebrafish compared to mice (Prusky et al., 2000). Despite the similar discrimination index obtained in the two species, we observed that fish discriminated different shapes from those discriminated by mice. Nothing is known about the discrimination criterion used by mice and zebrafish. However, we only observed that the pairing of two shapes (for example, •-■ and c-•) was discriminated by zebrafish and mice while others (▲- ) were poorly discriminated from both animal species. The predictive validity of VORT was tested by investigating the effects of amnesic and pro-mnesic cholinergic drugs. Nicotine improved the mean discrimination index using poorly discriminated shapes and the nicotinic or muscarinic antagonists, mecamylamine and scopolamine, respectively, significantly reduced the same parameter when discriminated shapes were used. We can exclude the possibility that the improvement or the impairment obtained with cholinergic drugs was influenced by changes in general activity because the total number of crossed lines in the swimming activity test was not modified as previously reported using the same drugs (Braida et al., 2013a). As in rodents, nicotine is involved in zebrafish cognitive enhancement using a 3-chamber task (Levin and Chen, 2004). Nicotine significantly improved choice accuracy of zebrafish in a simple spatial discrimination learning task (Cognato et al., 2012; Eddins et al., 2009; Levin et al., 2006). The facilitating effect was slowed by mecamylamine
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 4. Effect of movement applied to discriminated shapes in VORT. Mean discrimination index (A), mean exploration time (B) and examples of movements (C) applied to the same (left) or different shapes (right). Data are expressed as mean ± S.E.M. N = 18/33 for each group. #p b 0.05 as compared to corresponding familiar shape T2 (Tukey's test).
Fig. 5. Effect of movement applied to poorly discriminated shapes in VORT. Mean discrimination index (A), mean exploration time (B) and examples of movements (C). During T1 the two identical shapes were presented having no movement (stationary shapes) or the same movement. During T2 a novel shape was presented without motion (stationary shapes) or with the same or different movement from T1. Data are expressed as mean ± S.E.M. N = 10 for each group. ****p b 0.01 as compared to stationary shapes; $ p b 0.05, $$$ p b 0.01 as compared to corresponding familiar shape (T2) (Tukey's test).
(Eddins et al., 2009) while scopolamine induced memory deficits in an inhibitory avoidance paradigm (Richetti et al., 2011). The adopted nicotine, scopolamine and mecamylamine doses, were similar to those used in a T-maze task (Braida et al., 2013a) and within the same range adopted in mice using VORT (Braida et al., 2013b). These results confirm the close similarity between the two species. Notably, sequence identities of nicotinic acetylcholine receptor (nAChR) subunits in both species were determined. Mouse-zebrafish nAChR orthology indicated a percentage of identity between 52 and 79% for the nAChR subunits (Ensembl Zv9 web site). It is important to note that, unlike other reports in which nicotine was dissolved in the water tank (Kedikian et al., 2013; Levin, 2011; Miller et al., 2013; Sackerman et al., 2010), we employed the IP route to control more precisely the amount of drug each fish received. It can be argued that handling the fish could generate a state of anxiety. In
our experiments we chose cold water rather than chemical anaesthesia in order to limit the effect of stress as much as possible. Accordingly, no increase of blood glucose levels, which is an index of stress in teleost fish, has been found under this kind of anaesthesia (Kinkel et al., 2010). In addition, minimal lethality was observed after ice-induced anaesthesia. Nicotine probably increased attention and enabled animals to distinguish very similar visual stimuli whose discrimination under normal circumstances was not easy. It is not surprising that after nicotine injection no further improvement was observed in the mean discrimination index using highly discriminated shapes. At least in mice, the lack of an effect, following nicotine treatment, might be due to a ceiling effect that did not allow further improvement in performance (Young et al., 2012). Accordingly, no further effect was shown in mice using the same task (Braida et al., 2013b).
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Notably, the present findings demonstrate, for the first time, that zebrafish can identify shapes with characteristic motion. Zebrafish are also able to discriminate the direction of motion (vertical, horizontal or oblique) independently from the presented shape information. More interestingly, attention was increased when motion was applied to stationary, poorly discriminated, shapes. Indeed, applying the same movement in both T1 and T2 the discrimination index increased, supporting the notion that motion is a powerful cue that makes the task more valuable for studying attention. This indicates that motion is a strong attentional cue causing a shift of visual attention towards the moving shapes. These findings are in line with those recently found in mice using 2D moving shapes (Braida et al., 2013b). There is evidence that motion may contribute to object recognition also in humans (Foster and Gilson, 2002) and in patients affected by neurodegenerative diseases such as Alzheimer (Poissonnet et al., 2012) and Parkinson's (Meppelink et al., 2009).
a future task automation that could minimize the variability due to manual scoring and making the test less time consuming. Until now, the only task suitable to measure sustained attention in zebrafish is 3CSRTT. However, it appears a complex and long-lasting task, not particularly adapted to rapidly screen new drugs. Thus, we feel that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and to predict the efficacy of pharmacotherapies for cognitive disorders.
Disclosure statement All authors disclose any actual or potential conflict of interest.
Acknowledgements 5. Conclusion Taken together our findings support the value of performing VORT in zebrafish to study attention and provide the basis for
The authors thank Alan Langus (SISSA/ISAS International School for Advanced Studies, Trieste, Italy) for the technical assistance to iPod generation of shapes.
Appendix A
Fig. A. Photo of VORT apparatus and pictures of different phases.
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
A new model to study visual attention in zebrafish Daniela Braida a,b, Luisa Ponzoni a,c, Roberta Martucci a, Mariaelvina Sala a,d,⁎ a
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy Fondazione IRCCS Don Gnocchi, Milan, Italy c Fondazione Fratelli Confalonieri, Milan, Italy d CNR, Institute of Neuroscience, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 14 March 2014 Accepted 14 March 2014 Available online xxxx Keywords: Cholinergic system Learning and memory Moving shapes Teleost Visual attention
a b s t r a c t The major part of cognitive tasks applied to zebrafish has not fully assessed their attentional ability, a process by which the nervous system learns, organizes sensory input and generates coordinated behaviour. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply a modified version of novel object recognition test named virtual object recognition test (VORT) using 2D geometrical shapes (square, triangle, circle, cross, etc.) on two iPod 3.5-inch widescreen displays, located on two opposite walls of the water tank. Each fish was subjected to a familiarization trial (T1), and after different time delays (from 5 min to 96 h) to a novel shape recognition trial (T2). A progressive decrease, across time, of memory performance, in terms of mean discrimination index and mean exploration time, was shown. The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index. Zebrafish discriminated different movements (vertical, horizontal, oblique) and the discrimination index increased significantly when moving poorly discriminated shapes were presented, thus increasing visual attention. Taken together these findings demonstrate that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and for predicting the efficacy of pharmacotherapies for cognitive disorders. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Zebrafish are providing an attractive model for behavioural studies. Although these fish are relative newcomers to studies of learning and memory (Sison et al., 2006), a number of studies revealed that they are capable of performing well in a range of learning tasks such as avoidance learning (Blank et al., 2009; Morin et al., 2013), olfactory conditioning (Braubach et al., 2009), shuttle box alternation learning (Pather and Gerlai, 2009), place conditioning (Eddins et al., 2009), appetitive choice discrimination (Bilotta et al., 2005), visual discrimination learning (Colwill et al., 2005), associative conditioning task (Luchiari and Chacon, 2013), active avoidance conditioning (Xu et al., 2007), alternation based spatial memory task (Williams et al., 2002) and even automated learning paradigm (Hicks et al., 2006). Sison and Gerlai (2011) have designed an associative learning task that was deliberately made to resemble a classical radial arm maze in which the traditional cue (food reward) was replaced by the sight of conspecifics. Parker Abbreviations: VORT, Virtual Object Recognition Test; 2D, bidimensional; T1, Familiarization trial; T2, Recognition Trial; IP, Intraperitoneally; 3D, Tridimensional; NOR, Novel Object Recognition Test; WT, Wild Type; NIC, Nicotine; SCOP, Scopolamine; MEC, Mecamylamine. ⁎ Corresponding author at: Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy. Tel.: + 39 02 50317042; fax: +39 02 50317036. E-mail address: [email protected] (M. Sala).
et al. (2012) have developed a 3-choice serial reaction time task (3CSRTT) in which zebrafish could learn to perform a complex operant task, which requires more than 20 days, similar to that developed for rodents to test sustained attention and impulsivity. More recently, a new T-maze task has been set up (Braida et al., 2013a). Zebrafish were trained to reach a reservoir and stay for at least 20 s. The running time difference between the first and the second trial was calculated as a measure of memory of the spatial location of reward. In these conditions nicotine enhanced memory while scopolamine impaired it. As recently reported by Echevarria et al. (2011), apart from the 3CSRTT, the majority of the above mentioned tasks have not fully assessed attentional ability, a process by which the organism learns and the nervous system receives or acquires sensory input and generates coordinated behaviour of animals (Bushnell, 1998). Cognitive impairment is a core symptom of neuropsychiatric and neurologic disorders. Attentional deficit has been considered to be one of the most consistent areas of impairment associated with psychiatric and neurodegenerative diseases (Kelip et al., 2008; Perry et al., 2000) as Parkinson's disease, Alzheimer, attention deficit hyperactivity, stroke, epilepsy (Biederman et al., 2006; Rubia et al., 2005; Sahakian et al., 1988; Stretton and Thompson, 2012) and schizophrenia (Lyon et al., 2012). Attention includes a series of cognitive processes and disorders related to attention may underlie cognitive dysfunctions. The novel object recognition (NOR) test evaluates an animal's attention that is elicited by the presentation of novel stimuli. Interest in NOR
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Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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is very recent and a large proportion of the literature on cognition has been dedicated to object and visual recognition in humans, pigeons and primates (Spetch et al., 2006; Wallis and Bülthoff, 1999). The principal advantage of the NOR test is the rapid testing sequence and no necessary training other than the initial exposure session. In a recent paper we created a modified version of NOR, named virtual object recognition test (VORT), in mice where the 3D objects were replaced with virtual stationary or moving geometrical 2D shapes presented on iPod screens (Braida et al., 2013b). This test, as that used in humans, is predominantly based on visual stimuli. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply VORT to fish either using stationary or moving 2D shapes. It is known that cortical acetylcholine release has been implicated in novelty-induced arousal, attention, the encoding of novel stimuli and memory consolidation (Acquas et al., 1998; Sarter and Bruno, 2000). Thus, to test the task validity, different cholinergic drugs were injected intraperitoneally (IP) to fish.
The shapes were looped on a 3rd generation iPod Touch (Apple) through iTunes for the duration of the experiment (320 pixels horizontal axis and 480 pixels vertical axis). The luminosity of the screens was constant across the two screens and testing sessions. After T1 the fish returned to the home tank. During T2 each fish was placed again in the central area after different time delays, from 5 min to 96 h, during which one of the two identical static familiar shapes was replaced with a novel one. The time delays and doses were chosen on the basis of our previous work, using the same task, in mice (Braida et al., 2013b). For dynamic cue the same or different shapes presented in T 1 moved in a different direction than in T1. Attention was paid to counterbalance the choice of the shapes. Within every time delay, all the pairing discriminated shapes were randomly tested. Shape recognition was manually scored with a stopwatch, by an experimenter blind to the treatment, in terms of exploration time whenever the zebrafish approached to the iPod area (10 cm) and directed its head toward the shape. All the fish were drug naive, and each fish was used only once. 9–11 fish per group were used.
2. Materials and methods
2.3. Drug and treatment
2.1. Animals
Body weight was measured as previously described (Braida et al., 2007). Fish were removed from their home tank using a net and placed in a container, filled with tank water, positioned on a digital balance. Zebrafish weight was determined as the weight of the container plus the fish minus the weight of the container before the fish was added. The mean of three measurements was recorded. All the drugs were injected IP in a volume of 2 μl/g using a Hamilton syringe, according to Braida et al. (2013a). All drugs were dissolved in sterile saline (0.9%) and were prepared fresh daily. The drugs were: nicotine bi-tartrate salt (Sigma–Aldrich, St.Louis, MO, USA), (0.02 mg/kg) given 20 min before the test; scopolamine hydrobromide (Sigma–Aldrich, St. Louis, MO, USA) (0.025 mg/kg) given 20 min before the test; and mecamylamine hydrochloride (Sigma–Aldrich, St. Louis, MO, USA) (0.1 mg/kg) given 30 min before the test. Control groups received sterile saline solution (0.9%) which was given 20 or 30 min before T1. The range of doses of nicotine and of antagonists were chosen on the basis of their activity on T-maze task and on the basis that they did not modify swimming behaviour (Braida et al., 2013a).
Adult short-finned wild-type (WT) zebrafish (Danio rerio) (0.4–1 g) of heterogeneous genetic background were obtained from a local aquarium supply store (Aquarium Center, Milan, Italy). Zebrafish were 6–12 months of age and were 3–4 cm long. In all experiments, the sex ratio of zebrafish was approximately 50–50%. Males and females were identified as previously reported (Braida et al., 2012). Fish were kept at approximately 28.5 °C on a 14:10-h light/dark cycle. Behavioural testing took place during the light phase between 09:00 and 14:00 h. Tank water consisted of deionized water and sea salts (0.6 g/10 L of water; Instant Ocean, Aquarium Systems, Sarrebourg, France). Approximately 30 adult fish were maintained in 96 L home tanks (75 cm long, 32 cm wide and 40 cm high) provided with constant filtration and aeration. Animals were acclimated for at least 2 weeks before the experiments. Fish were fed daily with brine shrimp and flake fish food (tropical fish food, Consorzio G5, Italy). The experimental protocol was approved by the Italian Governmental Decree No. 18/2013. All efforts were made to minimize the number of animals used and their discomfort.
2.4. Statistical analysis 2.2. Virtual object recognition test (VORT) A transparent Plexiglas tank (filled with tank water at a level of 10 cm) was used in VORT. The apparatus (Fig. A) was characterized by a rectangular environment (70 cm × 30 cm × 10 cm). After a week habituation to the apparatus, each fish was restricted for 5 min in a 20 cm central area delimited by two opaque barriers to visually isolate the stimuli areas where two identical white geometrical shapes on a black background were shown on two iPod 3.5-inch widescreen displays located externally to the opposite 10 cm wide walls. Then, each fish was subjected to a familiarization trial (T1), and after different time delays to a novel shape recognition trial (T2). Both T1 and T2 consisted of a 10 min session during which two identical white geometrical shapes on a black background were shown on two iPod screens. For static cue, shapes were simple geometrical shapes (square, triangle, circle, cross, etc.) with equal surface (2.5 cm2). A complete list is given in Fig. 2C. For dynamic cue, the same above shapes moved horizontally, vertically or diagonally (distance 320 px) at a constant speed of 120 px/s. The videos were created in Adobe Flash, with a frame size of 320 pixels × 480 pixels, with a rate of 30 frames per second. The videos were encoded with a video encoder H264 (Apple), a software program that converts information written in one format or code to a different one. Usually, this is carried out in order to standardize information, improve speed and security, and reduce the file in size which then saves space.
All data were expressed as mean ± S.E.M. Pair-wise comparisons were assessed with Student's t-test. Different groups were assessed by one-way analysis of variance (ANOVA) for multiple comparisons followed by Tukey's post-hoc test. Data were expressed as discrimination index [(time spent exploring novel shape − time exploring familiar shape) / (time spent exploring novel shape + time exploring familiar shape)], as previously described in mice (Braida et al., 2013b). Data from fish receiving saline 20 or 30 min before T1 were pooled after making sure that there was no statistical difference between the two groups. The level of significance was taken as p b 0.05. All statistical analyses were done using software Prism, version 6 (GraphPad, San Diego, CA, USA). 3. Results 3.1. Different time delays progressively decrease memory performance In Fig. 1 the effects of different time delays on VORT, in terms of discrimination index (Fig. 1A) and the exploration time respectively (Fig. 1B), are shown. One-way ANOVA showed a significant difference among groups in delay time (from 5 min to 96 h) (F(3,36) = 7.46; p b 0.0005). Post-hoc analysis revealed that the mean discrimination index, evaluated at 96 h, was significantly lower than that evaluated at the remaining intervals. Concerning the exploration time, one-way ANOVA revealed a significant difference among the groups (F(9,90) =
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 1. Performance evaluated in VORT (A–B) at increasing time delays. Mean discrimination index (A) and mean exploration time (B) evaluated when zebrafish were presented highly discriminated shapes. Data are expressed as mean ± S.E.M. N = 9–11 for each group. § p b 0.05, §§ p b 0.01 as compared to 96 h group; *p b 0.05, **p b 0.01, ***p b 0.001 as compared to corresponding familiar exploration time (Tukey's test).
6.21; p b 0.0001). Post-hoc analysis showed that the time spent to explore the new shape was higher than the familiar one at all the tested time delays except for 96 h. The best performance was obtained at the inter-trial of 5 min; thus, such delay was adopted in the following experiments. 3.2. Manipulation of cholinergic system alters the ability of fish to discriminate shapes On the basis of previous findings obtained in mice (Braida et al., 2013b) a number of shapes were tested for their ability to be discriminated by fish. Fig. 2C shows which shapes were easily discriminated (left) and which shapes were not (right). The obtained mean discrimination indices were significantly different (t = 5.47, p b 0.0001) (Fig. 2A). Accordingly, when using highly discriminated shapes more time was spent exploring the novel shape than the familiar one (F(3,36) = 3.53, p = 0.001) (Fig. 2B). In contrast, a similar mean exploration time for the familiar and the novel shapes, for the poorly discriminated shapes, was obtained (F(3,36) = 1.53, p = 0.81). The effects of different cholinergic treatments on VORT are shown in Fig. 3. We first investigated whether memory performance could be improved by nicotine treatment using shapes that were difficult to discriminate or whether further improved using highly discriminated shapes. Then we investigated whether treatment with scopolamine or mecamylamine impaired memory. Nicotine increased mean discrimination index compared to saline group (t = 3.55, p = 0.002). When fish were given different cholinergic antagonists there was a significant difference in the discrimination index among groups (F(3,36) = 18.17, p b 0.0001). Post-hoc tests revealed that, when discriminated shapes
Fig. 2. Performance evaluated in VORT using highly discriminated or poorly discriminated shapes. Mean discrimination index (A), mean exploration time (B) and examples of highly discriminated (left) and poorly discriminated (right) shapes used for the experiment (C). Data are expressed as mean ± S.E.M. N = 9–11 for each group. ****p b 0.0001 as compared to discriminated shapes (Student's t-test); @@@ p b 0.001 as compared to corresponding familiar shapes (T2) (Tukey's test).
were presented to zebrafish, both scopolamine and mecamylamine decreased mean discrimination index in comparison with saline, while treatment with nicotine did not further improve this parameter. One-way ANOVA revealed a significant difference among groups in the mean exploration time when poorly discriminated shapes were presented to the group of animals treated with nicotine (F(3,36) = 3.14, p = 0.003). Post-hoc tests revealed that, during the T2 phase, the mean exploration time of the novel shape was higher than the familiar one (Fig. 3C). When discriminated shapes were presented, one way ANOVA revealed a significant difference in the exploration time of zebrafish treated with saline (F(3,36) = 2.63, p = 0.0008) and nicotine (F(3,36) = 3.14, p = 0.003 (Fig. 3D). Tukey's test indicated that zebrafish spent more time exploring novel than familiar shape in these groups. 3.3. Zebrafish are able to discriminate different movements applied to shapes and the application of movement increases attention In Fig. 4 the effect of movement applied to discriminated shapes on VORT is reported. When a novel movement was applied during T2 to the same or different new shapes (Fig. 4C), a similar discrimination
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 3. Effect of different cholinergic drugs on mean (±S.E.M.) discrimination index (A and B) and exploration time (C and D) evaluated in VORT. Performance was assessed using poorly discriminated (left) and highly discriminated (right). Nicotine (NIC) (0.02 mg/kg) or scopolamine (SCOP) (0.025 mg/kg) was injected IP 20 min before T1; mecamylamine (MEC) (0.1 mg/kg) was injected IP 30 min before T1. Saline = pool of fish receiving saline 20 or 30 min before T1. N = 10 for each group. §§p b 0.01 as compared to corresponding saline group (Student's t-test); °°°p b 0.001, °°°°p b 0.0001 as compared to corresponding saline and nicotine groups; **p b 0.01 as compared to corresponding familiar shape (T2) (Tukey's test).
index was obtained (t = 0.96 n.s.), indicating that zebrafish were able to distinguish different kinds of movements (vertical, horizontal, oblique). ANOVA showed that the exploration time (Fig. 4B) was significantly higher in T2 than in T1 between the two conditions when a new movement was introduced (same shapes: F(3,68) = 3.14, p b 0.03; different shapes: F(3,128) = 3.62, p b 0.01). We investigated also the possibility that fish could improve their performance when the movements were applied to poorly discriminated stationary shapes. As reported in Fig. 5A, the mean discrimination index increased significantly when moving shapes were presented (F(2,27) = 63.6; p = 0.0001). Post-hoc comparison revealed that the application of movement significantly increased the mean discrimination index either if the movement was different from T1 or it was the same. No difference was found in the mean exploration time when stationary shapes were presented (F(3,36) = 1.54; p N 0.05) (Fig. 5B). In contrast, there was a difference among groups in the mean exploration time when both different (F(3,36) = 8.13; p b 0.0001) and the same (F(3,36) = 4.56; p b 0.001) movements were applied to shapes. Post-hoc test revealed that during T2 there was a significant increase in the mean exploration time of novel shapes compared to the familiar ones, independently of whether the movement was changed or not (in Fig. 5C). 4. Discussion Our results demonstrate, for the first time, the possibility to evaluate selective attention in zebrafish using a modified version of the well established NOR task, which was applied to rodents to assess recognition memory for 3D objects (Ennaceur and Delacour, 1988). Zebrafish, submitted to VORT, at different inter-trial time delays, showed a progressive memory decay which was maximal when the delay of 96 h was applied. The obtained discrimination indices were comparable to
those previously found in mice submitted to the same task (Braida et al., 2013b). Notably, the mean exploration time was maintained to a higher level (more than 200 s) in zebrafish than in mice (less than 50 s) at all the tested time intervals. These differences could be due to the higher exploration activity of fish, compared to rodents, in the aquatic environment or to a different visual acuity. Measurement of visual acuity and contrast sensitivity, based on the OptoMotry device (Tappeiner et al., 2012), revealed a remarkable higher visual ability in adult zebrafish compared to mice (Prusky et al., 2000). Despite the similar discrimination index obtained in the two species, we observed that fish discriminated different shapes from those discriminated by mice. Nothing is known about the discrimination criterion used by mice and zebrafish. However, we only observed that the pairing of two shapes (for example, •-■ and c-•) was discriminated by zebrafish and mice while others (▲- ) were poorly discriminated from both animal species. The predictive validity of VORT was tested by investigating the effects of amnesic and pro-mnesic cholinergic drugs. Nicotine improved the mean discrimination index using poorly discriminated shapes and the nicotinic or muscarinic antagonists, mecamylamine and scopolamine, respectively, significantly reduced the same parameter when discriminated shapes were used. We can exclude the possibility that the improvement or the impairment obtained with cholinergic drugs was influenced by changes in general activity because the total number of crossed lines in the swimming activity test was not modified as previously reported using the same drugs (Braida et al., 2013a). As in rodents, nicotine is involved in zebrafish cognitive enhancement using a 3-chamber task (Levin and Chen, 2004). Nicotine significantly improved choice accuracy of zebrafish in a simple spatial discrimination learning task (Cognato et al., 2012; Eddins et al., 2009; Levin et al., 2006). The facilitating effect was slowed by mecamylamine
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Fig. 4. Effect of movement applied to discriminated shapes in VORT. Mean discrimination index (A), mean exploration time (B) and examples of movements (C) applied to the same (left) or different shapes (right). Data are expressed as mean ± S.E.M. N = 18/33 for each group. #p b 0.05 as compared to corresponding familiar shape T2 (Tukey's test).
Fig. 5. Effect of movement applied to poorly discriminated shapes in VORT. Mean discrimination index (A), mean exploration time (B) and examples of movements (C). During T1 the two identical shapes were presented having no movement (stationary shapes) or the same movement. During T2 a novel shape was presented without motion (stationary shapes) or with the same or different movement from T1. Data are expressed as mean ± S.E.M. N = 10 for each group. ****p b 0.01 as compared to stationary shapes; $ p b 0.05, $$$ p b 0.01 as compared to corresponding familiar shape (T2) (Tukey's test).
(Eddins et al., 2009) while scopolamine induced memory deficits in an inhibitory avoidance paradigm (Richetti et al., 2011). The adopted nicotine, scopolamine and mecamylamine doses, were similar to those used in a T-maze task (Braida et al., 2013a) and within the same range adopted in mice using VORT (Braida et al., 2013b). These results confirm the close similarity between the two species. Notably, sequence identities of nicotinic acetylcholine receptor (nAChR) subunits in both species were determined. Mouse-zebrafish nAChR orthology indicated a percentage of identity between 52 and 79% for the nAChR subunits (Ensembl Zv9 web site). It is important to note that, unlike other reports in which nicotine was dissolved in the water tank (Kedikian et al., 2013; Levin, 2011; Miller et al., 2013; Sackerman et al., 2010), we employed the IP route to control more precisely the amount of drug each fish received. It can be argued that handling the fish could generate a state of anxiety. In
our experiments we chose cold water rather than chemical anaesthesia in order to limit the effect of stress as much as possible. Accordingly, no increase of blood glucose levels, which is an index of stress in teleost fish, has been found under this kind of anaesthesia (Kinkel et al., 2010). In addition, minimal lethality was observed after ice-induced anaesthesia. Nicotine probably increased attention and enabled animals to distinguish very similar visual stimuli whose discrimination under normal circumstances was not easy. It is not surprising that after nicotine injection no further improvement was observed in the mean discrimination index using highly discriminated shapes. At least in mice, the lack of an effect, following nicotine treatment, might be due to a ceiling effect that did not allow further improvement in performance (Young et al., 2012). Accordingly, no further effect was shown in mice using the same task (Braida et al., 2013b).
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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Notably, the present findings demonstrate, for the first time, that zebrafish can identify shapes with characteristic motion. Zebrafish are also able to discriminate the direction of motion (vertical, horizontal or oblique) independently from the presented shape information. More interestingly, attention was increased when motion was applied to stationary, poorly discriminated, shapes. Indeed, applying the same movement in both T1 and T2 the discrimination index increased, supporting the notion that motion is a powerful cue that makes the task more valuable for studying attention. This indicates that motion is a strong attentional cue causing a shift of visual attention towards the moving shapes. These findings are in line with those recently found in mice using 2D moving shapes (Braida et al., 2013b). There is evidence that motion may contribute to object recognition also in humans (Foster and Gilson, 2002) and in patients affected by neurodegenerative diseases such as Alzheimer (Poissonnet et al., 2012) and Parkinson's (Meppelink et al., 2009).
a future task automation that could minimize the variability due to manual scoring and making the test less time consuming. Until now, the only task suitable to measure sustained attention in zebrafish is 3CSRTT. However, it appears a complex and long-lasting task, not particularly adapted to rapidly screen new drugs. Thus, we feel that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and to predict the efficacy of pharmacotherapies for cognitive disorders.
Disclosure statement All authors disclose any actual or potential conflict of interest.
Acknowledgements 5. Conclusion Taken together our findings support the value of performing VORT in zebrafish to study attention and provide the basis for
The authors thank Alan Langus (SISSA/ISAS International School for Advanced Studies, Trieste, Italy) for the technical assistance to iPod generation of shapes.
Appendix A
Fig. A. Photo of VORT apparatus and pictures of different phases.
Please cite this article as: Braida D, et al, A new model to study visual attention in zebrafish, Prog Neuro-Psychopharmacol Biol Psychiatry (2014), http://dx.doi.org/10.1016/j.pnpbp.2014.03.010
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