The endocannabinoid system and associative learning and memory in zebrafish.

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Research report

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The endocannabinoid system and associative learning and memory in zebrafish

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Tim Ruhl ∗ , Kirstin Moesbauer, Nadine Oellers, Gerhard von der Emde University of Bonn, Institute of Zoology – Department for Neuroethology and Sensory Ecology, Endenicher Allee 11-13, 53115 Bonn, Germany

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Zebrafish medial pallium is identified to be crucially involved in emotional associative learning and memory. Zebrafish medial pallium is known to contain high density of endocannabinoid receptor CB1. Acute activation/inactivation of CB1 has no effect on memory retrieval from associative memory in zebrafish. Chronic activation/inactivation of CB1 enhances or disrupts associative acquisition learning depending on the learning motivation.

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Article history: Received 29 October 2014 Received in revised form 11 March 2015 Accepted 27 April 2015 Available online xxx

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Keywords: Associative learning Memory Fish Endocannabinoid System Motivation Avoidance Telencephalon

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1. Introduction

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In zebrafish the medial pallium of the dorsal telencephalon represents an amygdala homolog structure, which is crucially involved in emotional associative learning and memory. Similar to the mammalian amygdala, the medial pallium contains a high density of endocannabinoid receptor CB1. To elucidate the role of the zebrafish endocannabinoid system in associative learning, we tested the influence of acute and chronic administration of receptor agonists (THC, WIN55,212-2) and antagonists (Rimonabant, AM-281) on two different learning paradigms. In an appetitively motivated two-alternative choice paradigm, animals learned to associate a certain color with a food reward. In a second set-up, a fish shuttle-box, animals associated the onset of a light stimulus with the occurrence of a subsequent electric shock (avoidance conditioning). Once fish successfully had learned to solve these behavioral tasks, acute receptor activation or inactivation had no effect on memory retrieval, suggesting that established associative memories were stable and not alterable by the endocannabinoid system. In both learning tasks, chronic treatment with receptor antagonists improved acquisition learning, and additionally facilitated reversal learning during color discrimination. In contrast, chronic CB1 activation prevented aversively motivated acquisition learning, while different effects were found on appetitively motivated acquisition learning. While THC significantly improved behavioral performance, WIN55,212-2 significantly impaired color association. Our findings suggest that the zebrafish endocannabinoid system can modulate associative learning and memory. Stimulation of the CB1 receptor might play a more specific role in acquisition and storage of aversive learning and memory, while CB1 blocking induces general enhancement of cognitive functions. © 2015 Published by Elsevier B.V.

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Associative learning is defined as the ability to acquire and consolidate the relationship between the occurrence of a particular sensory stimulus and a subsequent consequence, which can have an either positive or negative relevance [1,2]. Typically food rewards are positive consequences for associative learning, while electric shocks or noxious substances are applied for punishment or

∗ Corresponding author. Tel.: +49 228733751; fax: +49 228735556. E-mail address: [email protected] (T. Ruhl).

visceral discomforts [3,4]. Both types of associative learning have been investigated in the zebrafish (Danio rerio) [5–7] involving different sensory modalities. Often animals were trained in visual association tasks [8–11], but also olfactory [12] and gustatory [13] based associative learning was investigated. Developmental and hodological findings suggest that the medial pallium (Pm) represents an amygdala homologous structure in the teleost dorsal telencephalon [14–17]. This assumption is supported by lesion studies in goldfish (Carassius auratus). Destruction of Pm resulted in an impaired retention of a previously acquired strategy during active avoidance conditioning [18–20]. Furthermore, mapping of neuronal activity by measuring expression of c-fos mRNA

http://dx.doi.org/10.1016/j.bbr.2015.04.046 0166-4328/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Ruhl T, et al. The endocannabinoid system and associative learning and memory in zebrafish. Behav Brain Res (2015), http://dx.doi.org/10.1016/j.bbr.2015.04.046

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revealed that Pm serves as a brain center for emotionally guided behavior in zebrafish [21]. Hence, the Pm is an essential structure for associative learning. The cannabinoid receptor 1 (CB1) represents the major target for endo- and exogenous cannabinoids in the central nervous system of vertebrates [22,23]. Lam et al. [24] investigated the distribution of CB1 mRNA expression in the brain of adult zebrafish. On the level of the dorsal telencephalon, the Pm showed most intense staining compared to other regions. This led to the assumption that the endocannabinoid system is crucially involved in Pm related processes like the fish’s emotional state or associative memory. Actually, acute treatment of zebrafish trained in a color discrimination paradigm with the CB1 agonist THC (9 tetrahydrocannabinol) did not affect memory retrieval, while a dose dependent anxiolytic effect was suggested [25]. The present work aimed to determine the influence of acute and chronic treatment with CB1 agonists and antagonists on behavioral performance, associative learning and memory retrieval in zebrafish trained either in an appetitively motivated color discrimination task or in an aversively motivated active avoidance paradigm in a fish shuttle-box. 2. Materials and methods 2.1. Animals Zebrafish (n = 132; D. rerio, AB wild type strain, 30–40 mm body length, one year of age, both sexes) were bred in-house and accomodated communally (maximum n = 100) in 200-l aquaria with aerated and filtered water at 25–27 ◦ C, a substrate of sand and different hiding places (plants, stones and potsherds). Animals were subjected to a 12:12 h light/dark cycle and maintained on a daily diet of pellet fish food (Sera, Heinsberg, Germany). Before beginning of learning Experiment 1 (see Section 2.3), participating individuals (n = 85) were not fed for four days. During the experiments, animals did not get extra food besides rewards from the tests. Zebrafish (n = 47) taken for learning experiment 2 (see Section 2.4), were not food deprived. Before beginning of the experiments, zebrafish were transferred to the test laboratory and were housed in groups of five animals in 12-l aquaria equipped with a heater and a water filter. Half of the aquariumwater was exchanged once a week. The experiments started after an acclimatization period of three days. All fish were drug naive prior to the experiments. The experiments followed the guidelines of the animal welfare laws and were approved by the Animal Care and Use Committee of the state of North Rhine-Westphalia, Germany. 2.2. Drug application To check how activation or inactivation of cannabinoid receptors changes behavioral performances in the two learning tasks, zebrafish were administered the CB1 agonists THC (THC-Pharm GmbH, Germany) or WIN55,212-2 (Sigma–Aldrich, Germany), or the CB1 antagonists Rimonabant (THC-Pharm GmbH, Germany) or AM-281 (Tocris, UK) as described before [25]. For the drug treatment animals were swimming in a beaker containing 100 nM drugs in 2.5 liter of tap-water for one hour. To reach the desired concentration, 250 ␮l of a 1 mM stock solution (3.14 mg THC or 5.23 mg WIN55,212-2, 268.6 ␮l ethanol, 700 ␮l cremophor, 9 ml aquadest ; 4.6 mg Rimonabant or 5.57 mg AM-281, 300 ␮l ethanol, 700 ␮l cremophor, 9 ml aquadest ) was added to the aquarium water. Control animals were kept in tap-water containing the same amount of vehicle. Zebrafish were either tested for an acute effect and treated only once, or they were used for the investigation of chronic

pharmacological influences and received a daily treatment before each training. 2.3. Experiment 1 – color discrimination learning The color discimination paradigm followed the same protocol as described earlier [25] and is here described only briefly: The set-up for color discrimination learning was a 50-l aquarium that was divided into three compartments. A central starting chamber was separated from two choice-compartments by gray PVC-walls, each containing a central lockable door. At the outer sides of the choice-compartments, removable colored foils were fixed to the aquarium walls. Each foil was illuminated by a light bulb from behind, coloring the respective choice compartment and thus providing a color stimulus for learning. The light intensities of colors in the two choice-compartments were set equally to 500 lx. In a two-alternative choice task, zebrafish learned to associate a green color stimulus (S+ ) with a food reward. First, the animals were habituated to the unknown environment for two days. All individuals of one group were placed together in the starting chamber for 30 min, with both doors open and with two identical green colorstimuli. The group could freely explore the aquarium. Each time a door was passed, the animals were food rewarded with small pieces of blood worms. On the following two days, animals were set individually into the starting chamber, while the procedure was otherwise the same as on the previous days. After the habituation period, zebrafish were trained individually by releasing single fish into the starting chamber. After two minutes of acclimatization, the first trial started by opening both gates simultaneously. Fish learned to discriminate between two colors (green and red) by swimming to the color defined as positive (green: S+ ) to get a food reward. If a fish swam to the negative color (red: S− ), it was not rewarded and returned to the starting chamber. Before a new trial started, the two colored foils for S+ and S− were re-arranged according to a pseudorandom schedule [26]. The same color combination of S+ and S− was used during each training day. Daily sessions consisted of 4–8 trials for each individual. Correct or wrong decisions and the time from opening the gates until the fish swam through one of the doors were noted for each trial. Once the group had reached the learning criterion of more than 70% correct choices on three consecutive days, tests with acute pharmacological treatment were performed (n = 14 THC test, n = 13 Rimonabant test). To investigate the effect of chronic pharmacological treatment on learning and memory, other groups of fish (n = 11 control, n = 11 vehicle, n = 8 THC, n = 9 WIN55,212-2, n = 10 Rimonabant, n = 9 AM281) were treated with the drugs before each test for 13 days. Fish were trained as decribed above for seven days followed by six days of reversal training, during which choice of the previously incorrect color (red) was now rewarded and the previously correct color (green) was now incorrect. The procedure of daily training and measurements were the same as that used in the previous discrimination learning. 2.4. Experiment 2 – active avoidance learning For active avoidance learning, single zebrafish were trained in a fish-shuttle box modified after Pradel et al. [5]. A 12-l tank was divided by an opaque barrier into two equal compartments. The barrier was 10 cm high and the water level was set at 12 cm to allow the animals to freely pass over the barrier from one compartment into the other. There was a red foil at the end of each tank wall illuminated by a white LED behind it. In each compartment at the long sides of the tank, there were two steel electrode plates (13 cm × 16 cm). The steel electrodes were connected to a shocker unit consisting of an accupulser (Model A310, World Precision Instruments, Sarasota, USA) and a stimulus generator (Model


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395, Wavetek, San Diego, USA). A mirror was mounted above the shuttle box to allow observation of the fish‘s behavior. The experimental paradigm consisted of the presentation of a neutral stimulus (red light) followed by mild electric shocks at regular 6 s intervalls (125 mA, 200 ms) serving as the unconditioned stimulus. The light signal was kept on during each trial of 30 s. Afterwards, the neutral stimulus and the unconditioned stimulus switched to the other side of the shuttle box. The daily training period lastet for 15 min and consisted of 30 trials per day. Fish had to associate the onset of the neutral stimulus with the upcoming unconditioned stimulus and to avoid the electric shocks by swimmimng to the other side of the tank within 6 s after the appearence of the red light. Before training, we determined the chance level of zebrafish shuttling into the other compartment within 6 s after onset of the red light stimulus without the consequent appearance of the unconditioned stimulus. The tested animals (n = 29) showed shuttling behavior in 29% of the trials. Because of this spontaneous shuttling frequency, we defined the learning criterion as 45% avoidance reactions, which differed significantly from the chance level of 29% (Fisher’s Exact Test, SPSS 21, SPSS Inc., Chicago, USA). Before the experiment started, zebrafish were habituated to the unknown environment for two days. Fish were set as a group into the shuttle box without any neutral or unconditioned stimuli. The animals were free to swim in the tank and could explore the barrier as well as the two compartments. On the third day, animals were placed individually into the shuttle box and after an acclimatization period of one minute, training started. When the red light turned on, a fish could avoid the electric pulses by crossing the barrier before the first electric pulse (i.e. within 6 s). If it stayed in the compartment longer than 6 s, it could either perform an escape reaction by swimmimng to the other side during/after the first electric pulse or respond with freezing behavior and stay in the shock compartement. All different types of behaviors were noted. Once the group achieved the learning criterion of reaching more than 45% avoidance responses on three consecutive days, the tests with acute pharmacological treatment were performed (n = 15). To test for an effect of chronic treatment with THC or Rimonabant on learning and memory, fish (n = 6 vehicle, n = 5 Rimonabant, n = 6 THC) got daily treatment over the course of the experiment. The procedure of daily training and measures was the same as mentioned above.

All data from behavioral experiments were expressed as means and standard error of the mean (±). Data of individuals investigated were grouped in order to evaluate the behavioral performance for each day of testing and analyzed for normality using the Kolmogorov–Smirnov test, followed by one-way ANOVA or two-way ANOVA with repeated measures (SPSS 21, SPSS Inc., Chicago, USA). Post-hoc analyses were performed by Bonferroni-correction. Statistical significance was accepted if p < 0.05. 3. Results

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towards the open doors before entering one choice chamber. ANOVA detected a significant overall increase in number of correct choices (F(12,169) = 3.6, p < 0.001) and a decrease in decision latencies (F(12,714) = 6.6, p < 0.001) over the course of the experiment for the THC group. The Rimonabant group started at a higher level in number of correct choices, initially swimming in 70% to S+ . Because of this, ANOVA did not indicate a significant increase in number of correct choices (F(9,117) = 1.5, p = 0.1), while the choice latencies significantly decreased (F(9,484) = 2.0, p < 0.05) during this period of the experiment. On days seven and eight, the pharmacological tests were performed and on day eleven there was the vehicle control. At this point, zebrafish had reached about 80% correct choices and needed 4–6 s for a decision. On the days of acute THC, Rimonabant or vehicle administration, the number of correct choices or the decision latencies did not change significantly compared to the days before or after, indicating that the treatments had no significant effects on fish behavior (Bonferroni, p = 1.0). 3.1.2. Acute treatment – Experiment 2 Analog results were obtained in the active avoidance learning experiments, during which zebrafish learned to associate a red light stimulus with a mild punishment and to shuttle between two compartments for avoiding an electric shock. During the habituation period, zebrafish were active and shuttled between the two compartments without any sign of fear. On the first two days of acquisition training however, the animals reacted with freezing (immobility) to the occurrence of the electric pulses. This is a common response of fish after being electrically stimulated and lasted for a period of 5–10 s before the animals started to show escape reactions [27]. Hence, they often stayed at one side of the fish shuttle-box and showed avoidance responses in less than 20% of the trials (Fig. 2), thus performing even lower than the designated chance level (29%). On day three, the number of avoidance reactions constantly started to rise to over 50%, finally surpassing the learning criterion (Fig. 2). ANOVA indicated a significant increase in avoidances (F(17,236) = 6.9, p < 0.001) over the course of the experiment. Pharmacological treatments were done on days 12, 14 and 18. Neither THC or Rimonabant application nor vehicle control had a significant effect on behavioral performance of the zebrafish in the shuttle-box compared to the days before and after (Bonferroni, p = 1.0) (Fig. 2).

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2.5. Analyses and statistics

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The results of the associative color discrimination experiment indicated that zebrafish of both groups (THC and Rimonabant groups) developed a strong preference for swimming to S+ after several days of training. During acquisition, the number of correct choices increased while the choice latency decreased from day one until day six (Fig. 1A and B). In this experimental phase, the animals inspected the two options by repetitively swimming

3.1.3. Chronic treatment – Experiment 1 Next we tested the effect of chronic treatment with CB1 agonist and antagonist on learning and memory using the same learning paradigms as described above. In the color discrimination experiment, five of the six test-groups showed successful choice preference for S+ during acquisition (days 1–7), only animals treated with CB1 agonist WIN failed to solve the color association task. During reversal learning (days 8–13) we found significant behavioral impairments in groups treated with CB1 agonists, while both CB1 antagonists tested significantly facilitated solving the memory task (Fig. 3). Daily pharmacological treatment with vehicle had no effect on color association of the tested zebrafish compared with untreated controls (Fig. 3A). ANOVA with repeated measures showed that number of correct choices was not significantly different between control and vehicle group either for acquisition period (Fgroup (1,18) = 0.01, p = 0.8; Fdays (6,108) = 3.4, p < 0.01; Fgroup × days (6,108) = 2.0, p = 0.07) or for reversal learning (Fgroup (1,20) = 0.3, p = 0.6; Fdays (5,100) = 1.0, p = 0.4; Fgroup × days (5,100) = 1.1, p = 0.4).


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Fig. 1. Influence of acute treatment on memory reversal in Experiment 1. Learning curves of two zebrafish groups tested in a two-alternative choice task for color association. One group (n = 14, black) was taken for acute THC and vehicle treatment, second group (n = 13, gray) served for acute Rimonabant administration. The percentage of correct choices (A) and choice latency for decision making (B) are plotted versus the days of the experiment. Data are mean values ± SEM. Arrows denote days of acute pharmacological Q4 treatment: red – THC, green – Rimonabant, gray – vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308

However, chronic treatment with the two agonists THC and WIN led to significant differences in learning behavior of treated animals (Fig. 3B). ANOVA with repeated measures determined that the number of correct choices differed significantly between the groups during acquisition (Fgroup (2,24) = 22.43, p < 0.001; Fdays (6,144) = 1.4, p = 0.2; Fgroup × days (12,144) = 0.7, p = 0.8). Post hoc analyses using the Bonferroni correction revealed that application of THC significantly improved behavioral performance over that of the control group (p = 0.001) and the WIN group (p < 0.001), and there was a significant difference between the control and the WIN group (p < 0.05), which was the only group that failed to acquire a color association. In addition, animals treated with WIN only showed increased thigmotaxis as they preferred to swim along the walls of the starting chamber before they chose to swim into one choice compartment. All the other fish tested swam at the center of the starting chamber, shuttling from between the walls.

Groups treated with both CB1 antagonists showed superior acquisition learning over the control group (Fig. 3C). ANOVA with repeated measures determined that the number of correct choices differed significantly between the three groups (Fgroup (2,26) = 8.18, p < 0.01; Fdays (6,156) = 2.4, p < 0.05; Fgroup × days (12,156) = 0.8, p = 0.7). Post hoc analyses after Bonferroni found that application of Rimonabant as well as AM-281 significantly improved behavioral performances over that of the control group (p = 0.01), while there was no significant difference between the treatment with both antagonists (p = 1.0). Starting of reversal learning made the WIN group achieve more correct choices than the control and the THC group, but this effect certainly was based on the fact that animals treated with WIN did not solve the original associative memory task before; their behavioral performance stayed around the 50% chance level for the whole duration of the experiment (Fig. 3B). During reversal


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Fig. 2. Influence of acute treatment on memory retrieval in Experiment 2. Avoidance learning of zebrafish (n = 15) tested in a fish shuttle-box. The percentage of avoidances is shown during the course of the experiment. Data are mean values ± SEM. Acute vehicle control (gray) was tested on day 12, THC (red) administration on day 14 and Rimonabant (green) on day 18. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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learning, the THC group performed at a very low level and did not improve its performance. These animals showed significantly fewer correct responses than the control group. For reversal learning, ANOVA with repeated measures found that the number of correct choices differed significantly between THC, WIN and control groups (Fgroup (2,23) = 13.4, p < 0.001; Fdays (5,115) = 1.2, p = 0.3; Fgroup × days (10,115) = 1.3, p = 0.3). A post hoc test after Bonferroni showed that THC significantly impaired reversal learning compared to the WIN (p < 0.001) and the control group (p < 0.05), while behavioral performance of controls differed significantly from that of WIN treated animals (p < 0.05). In contrast, the groups treated with both antagonists achieved significantly more correct choices than the control group during reversal learning. ANOVA with repeated measures found that the number of correct choices differed significantly between Rimonabant, AM-281 and control group (Fgroup (2,25) = 12.1, p < 0.001; Fdays (5,125) = 4.4, p < 0.001; Fgroup × days (10,125) = 1.2, p = 0.3). The Bonferroni post hoc test revealed that antagonists significantly improved reversal learning over the control group (p < 0.01), while there was no difference between the behavioral performance of Rimonabant and AM-281 treated animals (p = 1.0).

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experiment. Only the THC group failed to achieve the learning criterion. The animals did not show a clear increase in avoidance responses over the course of the experiment. Instead the fish appeared every day as if they were confronted with the behavioral paradigm for the first time. If sometimes an animal showed a high number of avoidances on one day, the same individual failed to maintain this high performance and achieved only a low number of avoidances on the following day. ANOVA with repeated measures after Greenhouse–Geisser correction showed that the number of avoidances differed significantly between the THC, Rimonabant and control groups (Fgroup (2,20) = 4.6, p < 0.05; Fdays (5.3,105.5) = 6.5, p < 0.001; Fgroup × days (10.5,105.5) = 1.5, p = 0.1). Post hoc test after Bonferroni revealed that Rimonabant significantly improved behavioral performance in comparison to the group treated with THC (p < 0.05), while there was no significant difference between the control and the THC groups (p = 0.8). The difference between the Rimonabant and the control group was not significant but showed strong tendency (p = 0.07).

4. Discussion

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In contrast to color association learning, chronic pharmacological treatment influenced active avoidance learning in a different manner. In comparison to untreated controls, daily treatment with vehicle solution mildly affected learning behavior of the zebrafish (Fig. 4A). The control group performed slightly better than the vehicle group, but ANOVA with repeated measures after Greenhouse–Geisser correction determined that the number of avoidances did not differ significantly between the two groups (Fgroup (1,18) = 1.3, p = 0.3; Fdays (4.8,86.2) = 10.0, p < 0.001; Fgroup × days (4.8,86.2) = 1.1, p = 0.39). In contrast, chronic treatment with THC or Rimonabant significantly affected learning and memory in the fish shuttle-box (Fig. 4B). All three groups initially achieved low numbers of avoidance responses. While the control group improved its performance continuously, reaching the learning criterion after day six, the Rimonabant group showed faster learning, as the animals reached the learning criterion already on the second day and stayed on a high level until the end of the

This study examined the effects of acute and chronic treatment with cannabinoid receptor 1 (CB1) agonists (THC, WIN55,212-2) and antagonists (Rimonabant, AM-281) on appetitively and aversively motivated associative learning and memory functions in zebrafish. The results of the tests of acute pharmacological treatment after acquisition showed that neither the performance in the food rewarded color discrimination task nor in the fish shuttle-box was disrupted by THC or Rimonabant. This indicates that retrieval from associative memory once established appears to work rather independently of the endocannabinoid system in the zebrafish. In contrast, chronic treatment with CB1 antagonists and agonists had an effect on both learning tasks. In the color association task, the two agonists had opposite effects on color association: treatment with THC improved color association while WIN impaired behavioral performance in this memory task. The two CB1 antagonists Rimonabant and AM-281 both improved acquisition and facilitated reversal learning in the color association task. In the active avoidance paradigm, chronic treatment with the CB1 antagonist


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Fig. 4. Influence of chronic treatment on acquisition learning in Experiment 2. (A) Avoidance learning of an untreated control group (n = 15, black) and of zebrafish after daily treatment with vehicle (n = 6, gray). (B) Number of avoidances of the control group (black), the Rimonabant group (n = 5, green) and the THC group (n = 6, red) over the days of testing. Data are mean values ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3. Influence of chronic treatment on acquisition and reversal learning in Experiment 1. (A) Number of correct choices of untreated control group (n = 11, black) and zebrafish after daily vehicle treatment (n = 11, gray) trained in a color discrimination paradigm. (B) Learning curves of control group (black), THC (n = 8, red) and WIN (n = 9, blue) treated animals. (C) Learning curves of control group (black), Rimonabant (n = 10, green) and AM-281 (n = 9, brown) treated animals. Acquisition training lasted for 7 days, reversal learning for the following 6 days. Data are mean values ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Rimonabant significantly improved learning behavior of zebrafish, while the receptor agonist THC impaired avoidance learning.

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Associative learning requires the acquisition of a causal and/or temporal relationship between either a stimulus driven behavior

(swimming to S+ ) and a subsequent positive or negative consequence, or between two stimuli, an unconditioned stimulus (US) and a neutral stimulus that during conditioning becomes a conditioned stimulus (CS). In the case of Experiment 1, the color discrimination paradigm, swimming to the green color (S+ ) was associated with a food reward, while the red color was avoided because it was associated with no reward. In contrast, during active avoidance conditioning of Experiment 2, a mild electric shock served as the US, while the red light stimulus was the CS. Knowledge about the neuronal substrates for associative learning in fish originates mostly from lesion studies. Especially destruction of the medial pallium (Pm) in the dorsal telencephalon of teleosts impaired emotional learning, e.g. taste aversion learning [13]. Furthermore, lesions of the Pm resulted in the disrupted retention of conditioned avoidance responses (CAR) in a shuttlebox [18]. Analyses of gene expression and hodological as well as neurophysiological findings strongly suggest that among pallial areas, the Pm in fish is functionally homologous to the mammalian amygdala, while the lateral pallium (Pl) represents a hippocampus homolog structure [16,21,28–30]. As found for the amygdala and the hippocampus, Pm and Pl of the zebrafish telencephalon have a high density of CB1 [24,31–33]. Nevertheless, neither acute


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activation nor acute inactivation of CB1 in our zebrafish influenced retrieval from associative memory in both learning tasks at the concentrations tested (see also [25]). These results differ partly from the results of comparable studies conducted with mammals. In appetitively motivated learning tasks, i.e. in rewarded conditioning experiments such as our Experiment 1, similar results were obtained with mammals. Once a rewarded visual or olfactory association was established in rodents, it appeared to be unaffected by activation or inactivation of the endocannabinoid system [34,35]. In contrast, the results we obtained in the Experiment 2 differ from those obtained with mammals. While acute Rimonabant treatment did not have any effect on memory retrieval of mice in a shuttle-box [36], acute THC administration with different doses (2.5–7.5 mg/kg) in rats, which were trained in a Pavlovianto-instrumental transfer avoidance procedure or in conditioned avoidance responding in a shuttle-box, significantly reduced the number of CAR [37–39]. Maybe, the different findings between zebrafish and mammals can be explained by a dose-dependency of CB1 activation. Cannabinoid receptor agonists such as THC often dose-dependently induce opposite behavioral effects [40,41] in mammals. CB1 located on GABAergic terminals are activated by lower concentrations of agonists than receptors located on glutamatergic terminals [42], which possibly leads to the dose-dependent control of GABAergic and glutamatergic neurons on retrieval of CAR [43]. It follows that THC exerts a biphasic control of fear coping strategies, with lower and higher doses favoring active and passive responses, respectively [43]. Also in zebrafish, the number of CAR might decrease at THC doses higher than the 100 nM we used in this study, in a similar way as it was shown for rats: 20 mg/kg of cannabis resin with THC concentration of 17% did not inhibit CAR on the pole climbing apparatus, whereas application of 100 mg/kg induced complete suppression of CAR [44].

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Associative visual discrimination learning and discrimination reversal has been investigated in zebrafish [6,8,45–47]. Besides WIN, all groups tested in our experiments showed color association, as individuals could successfully discriminate between a rewarded and a non-rewarded color. As reported in the literature on mammals, CB1 antagonists can induce memory enhancement in several learning tasks [48], i.e. social recognition test [49], and spatial memory tasks [50,51]. In contrast, application of CB1 agonists such as THC cause memory impairments in mammals [52]. In our study, chronic treatment with the two CB1 agonists had different effects on color association. THC significantly enhanced acquisition learning, but WIN treated animals failed to associate a color with a food reward (Fig. 3B). This finding is paradox and suggests that the differences between both CB1 agonists might be caused by deviant pharmacokinetics and -dynamics. Unlike THC, WIN is a full agonist of CB1 with higher relative intrinsic activities and receptor affinity, which lies about 40-times higher than that of THC [53–56]. Thus, treatment with 100 nM WIN might be more effective at CB1 than 100 nM THC, causing a deficit in associative learning. Also, the influence of WIN on thigmotaxis could be interpreted as an elevated level of fear being responsible for impaired learning behavior [57,58], although WIN was found to act anxiolytically on zebrafish behavior [59]. Treatment with the other CB1 agonist THC led to opposite findings, namely that it significantly improved associative learning. Investigations on mammals reported that elevated levels of endogen CB1 ligand anandamide increased the acquisition rate in different learning paradigms [60–62]. Based on these findings, it has been proposed that a subtle modulation of CB1 activity might alter emotional or motivational states leading to improvement of memory acquisition. Hence, 100 nM THC improved

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acquisition learning could be a feature specifically attributed to the zebrafish endocannabinoid system, or it originated from a comparable nootropic effect that was found for elevated anandamide levels in mammals. Initiation of reversal learning by switching the reinforced color with the previously unrewarded alternative color decreased the number of correct choices in all groups. In a study by Colwill et al. [9], zebrafish trained in a color discrimination paradigm needed four days or even longer for reversal learning. Although a correction procedure was used, during which each incorrect trial was repeated with the incorrect choice option blocked, some animals needed more than seven days for discrimination reversal. In the present study, we did not use a comparable correction procedure and found that our zebrafish showed poor reversal learning, not even reaching the chance level of 50% correct choices after six days of training (except WIN, which failed to associate a color with a food reward and consequently did not show reversal learning). While untreated controls slightly improved their behavioral performance during this period, reversal learning in the THC group was significantly impaired. Even after six days of training, animals were still choosing the previously rewarded color in over 80% of trials. In contrast to this, animals treated with both CB1 antagonists were more flexible in their behavior and could better adapt to the altered scheme. They reached the learning criterion already after four days of training (Fig. 3C). These findings are in agreement with investigations done with mammals. Rimonabant facilitated the reversal of a previously learned olfactory discrimination in rats, while it was impaired by THC [35,63]. Lesion studies highlighted the orbitofrontal cortex (OFC) as neuronal substrate determining reversal learning in rats [64–67]. It was concluded that impaired discrimination reversal arose through a cannabinoid effect on OFC circuitry, being involved in impulse control and in updating affective associations between stimuli and reinforcement value [63,68]. Especially destruction of the ascending serotonergic projections from the medial dorsal thalamus to the OFC specifically impaired discrimination reversal in the marmoset (Callithrix jacchus) [69–71] and in rats [72], suggesting that ascending serotonergic input may be crucially involved in behavioral flexibility in mammals. Investigations on functional homology between mammalian and zebrafish brain anatomy defined the central zone (Pc) of the zebrafish dorsal pallial divisions as corresponding structure to the mammalian isocortex [16], which includes the above mentioned OFC. The posterior tuberculum represents a collection of thalamuslike relay stations in the zebrafish’s caudal diencephalon, with the quantitatively most important ascending projections to pallial areas including Pc [73–76]. Labeling experiments found serotonergic neurons and high expression of the CB1 receptor in the posterior tuberculum [24,77,78]. Hence, it might be possible that cannabinergic modulation of ascending serotonergic projections from the diencephalon onto the dorsal telencephalon were responsible for deficits in zebrafish reversal learning. 4.3. The endocannabinoid system and neurotransmitters of learning and memory Earlier studies on mammals showed that the endocannabinoid system selectively controls acquisition and storage of aversive memories. Especially the influences of CB1 elimination and receptor antagonists on extinction learning after fear conditioning were the topics of several investigations [79–81]. It was reported that genetic deletion or pharmacological inhibition of CB1 task-specifically disrupted extinction learning, being effective in aversive and ineffective in appetitive learning paradigms [82]. Consequently, the activation of CB1 by endo-/exogen receptor agonists could enhance extinction of aversive memories. In the present


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study, chronic treatment of zebrafish with Rimonabant exhibited enhanced acquisition of avoidance learning, while chronic activation of CB1 impaired this process. This finding is consistent with reports on mammals. Systemic administration of WIN prevented classical eyeblink conditioning in mice [83], while CB1 knockout animals showed a significant increase of CAR when trained in a shuttle-box [84]. Different hypotheses have been used to explain the molecular mechanisms determining cannabinoid induced impairments of learning and memory [85]. Electrophysiological approaches characterized CB1 agonists to disrupt long-term potentiation (LTP) in the hippocampus [86–88] and to modulate inhibitory networks in the amygdala [79], whereas antagonists had opposing effects [89]. Acetylcholine, GABA, and glutamate are three neurotransmitter systems through which cannabinoids could exert their effects in the hippocampus and the amygdala of mammals [31,33,85,90–94]. Principally similar to mammals, the zebrafish dorsal telencephalon contains glutamatergic as well as GABAergic neurons [95,96]. In zebrafish, glutamate was found to be essentially involved in single-trial inhibitory avoidance learning as well as in visual discrimination learning [97,98], while it was shown that piracetam, a GABA derivate, can improve spatial learning [99]. Furthermore, there are ascending cholinergic projections from the lateral nucleus of the ventral telencephalon onto the Pm and Pl [100,101] that are involved in learning and memory processes in zebrafish [102]. Further, electrical stimulation of Pl induces synaptic plasticity in Pm [30], suggesting that cannabinoid disturbance of Pl–Pm communication might inhibit learning processes similar to the mechanisms observed between hippocampus and amygdala in rats [103]. It might be possible that neurochemical mechanisms of cannabinoid effects on learning and memory in zebrafish resemble those hypothesized for mammals. Thus, zebrafish might be a promising model system for studying the physiology and pathophysiology of the endocannabinoid system and its involvement in learning and memory.

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Acknowledgement

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The drugs used in this study were generously provided by Andras Bilkei-Gorzo (University of Bonn – Institute of Molecular Psychiatry).

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References

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Explicit Associative Learning and Memory in Synesthetes and Nonsynesthetes.

Embryonic alcohol exposure impairs associative learning performance in adult zebrafish.

Effects of an acute seizure on associative learning and memory.

Molecular regulation of synaptogenesis during associative learning and memory.

Associative learning and timing.

Y-maze avoidance: an automated and rapid associative learning paradigm in zebrafish.

Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

The relationships between age, associative memory performance, and the neural correlates of successful associative memory encoding.

The endocannabinoid system and spermatogenesis.

Associative learning in invertebrates.

Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae.

Appetitive Olfactory Learning and Long-Term Associative Memory in Caenorhabditis elegans.

14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory.

Acute administration of THC impairs spatial but not associative memory function in zebrafish.

Associative learning alone is insufficient for the evolution and maintenance of the human mirror neuron system.

endovanilloid system and depression.

Nonbinary associative memory with exponential pattern retrieval capacity and iterative learning.

Early exposure to volatile anesthetics impairs long-term associative learning and recognition memory.

Associative concept learning in animals.

Individual differences in associative learning.

Associative memory cells: Formation, function and perspective.

Associative learning beyond the medial temporal lobe: many actors on the memory stage.

Associative memory cells: Formation, function and perspective.

The functions of contexts in associative learning.