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Discrimination conditioning. A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 45 s preceding the food reward release during the first conditioning trial of each conditioning day. B Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 60 s of the first memory test. C Time (in seconds, median and quartiles) spent by fish from paired group (n = 18, grey) in the CS+ (grey) and CS-(white) columns during the 60 s of the second memory test. D Time (in seconds, median and quartiles) spent by fish from unpaired group (n = 18) in the CS1 (green) and CS2 (red) columns
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The zebrafish is a laboratory species that gained increasing popularity the last decade in a variety of subfields of biology, including toxicology, ecology, medicine, and the neurosciences. An important phenotype often measured in these fields is behaviour. Consequently, numerous new behavioural apparati and paradigms have been developed for the ze...
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... semi-automated home-tank (Fig. 1A) was developed using easily accessible materials. We call it "semi"-automated, because as the description of procedures below shows, it still has a human handling component at the beginning and the end of each experimental day (Fig. S3). In addition, the protocols described in this manuscript represent classical conditioning, as they require CS-US association and not response-US association (operant conditioning) or multi-CSs relational learning (e.g. spatial learning). For example, the location of CS and US was irrelevant in our training methods. The experimental ...
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... were displayed using two RGB LED light, and food could be released from two homemade feeders (Fig. 1A, Fig. S1). These feeders were composed of a rotating servomotor (Micro servo 9G SG90, Miuzei, Japan) placed in a circular plastic container (#21470, Greenco, USA). The RGB LED light and homemade feeders were controlled via an Arduino type card ( Fig. S2, S3; Elegoo Carte Mega 2560, Elegoo, ...
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... Group:Colour effect: F = 0.11, df = 1, p = 0.74) and the generalization test (Colour effect: F = 2.26, df = 1, p = 0.14; Group:Colour effect: F = 2.28, df = 1, p = 0.14). For this reason, data were pooled for colour in this experiment. Nevertheless, performance of fish for each coloured-light is presented separately in the supplementary material (Fig. ...
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... conditioning, fish from the paired group significantly increased their time spent in the CS+ column across the trials as the training progressed, and consequently decreased the time spent in the CS-column (Fig. 3A, Table 1D). Although the time spent in each CS column did not significantly differ for the unpaired group, the performance of these fish significantly varied across training trials (Fig. 3D, Table ...
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... increased their time spent in the CS+ column across the trials as the training progressed, and consequently decreased the time spent in the CS-column (Fig. 3A, Table 1D). Although the time spent in each CS column did not significantly differ for the unpaired group, the performance of these fish significantly varied across training trials (Fig. 3D, Table ...
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... of the paired group spent significantly more time in the CS+ column than in the CS-during both memory test 1 (Fig. 3B, Table 1E) and test 2 (Fig. 3C, Table 1F). Similarly to the paired group, the performance of unpaired fish did not significantly change between the two memory tests (Test effect: Χ 2 = 0.05, df = 1, p = Fig. 2 Simple conditioning. A Time (in seconds, median and quartiles) spent by fish from paired group (n = 20, grey) and unpaired ...
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... of the paired group spent significantly more time in the CS+ column than in the CS-during both memory test 1 (Fig. 3B, Table 1E) and test 2 (Fig. 3C, Table 1F). Similarly to the paired group, the performance of unpaired fish did not significantly change between the two memory tests (Test effect: Χ 2 = 0.05, df = 1, p = Fig. 2 Simple conditioning. A Time (in seconds, median and quartiles) spent by fish from paired group (n = 20, grey) and unpaired group (n = 18, white) in the ...
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... during simple classical associative (CS-US) conditioning ( Fig. 2A). B Linear model results for the performance of fish during memory test (Fig. 2B). C Linear model results for the performance of fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model ...
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... performance of fish during memory test (Fig. 2B). C Linear model results for the performance of fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model ...
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... fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant ...
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... discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the ...
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... group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 45 s preceding the food reward release during the first ...
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... group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 45 s preceding the food reward release during the first conditioning trial of each conditioning day. B Time (in seconds, median and quartiles) spent by ...
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... group. *p < 0.05; **p < 0.01, ***p < 0.001; NS non-significant 0.81; Test:CS effect: F = 2.4, df = 1, p = 0.12). However, when analysing each test independently, fish from the unpaired group spent significantly more time in the CS1 (green) column than in the CS2 (red) one during test 1 (Fig. 3E, Table 1H), but this difference was absent in test 2 (Fig. 3F, Table ...
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... semi-automated home-tank (Fig. 1A) was developed using easily accessible materials. We call it "semi"-automated, because as the description of procedures below shows, it still has a human handling component at the beginning and the end of each experimental day (Fig. S3). In addition, the protocols described in this manuscript represent classical conditioning, as they require CS-US association and not response-US association (operant conditioning) or multi-CSs relational learning (e.g. spatial learning). For example, the location of CS and US was irrelevant in our training methods. The experimental ...
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... were displayed using two RGB LED light, and food could be released from two homemade feeders (Fig. 1A, Fig. S1). These feeders were composed of a rotating servomotor (Micro servo 9G SG90, Miuzei, Japan) placed in a circular plastic container (#21470, Greenco, USA). The RGB LED light and homemade feeders were controlled via an Arduino type card ( Fig. S2, S3; Elegoo Carte Mega 2560, Elegoo, ...
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... Group:Colour effect: F = 0.11, df = 1, p = 0.74) and the generalization test (Colour effect: F = 2.26, df = 1, p = 0.14; Group:Colour effect: F = 2.28, df = 1, p = 0.14). For this reason, data were pooled for colour in this experiment. Nevertheless, performance of fish for each coloured-light is presented separately in the supplementary material (Fig. ...
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... conditioning, fish from the paired group significantly increased their time spent in the CS+ column across the trials as the training progressed, and consequently decreased the time spent in the CS-column (Fig. 3A, Table 1D). Although the time spent in each CS column did not significantly differ for the unpaired group, the performance of these fish significantly varied across training trials (Fig. 3D, Table ...
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... increased their time spent in the CS+ column across the trials as the training progressed, and consequently decreased the time spent in the CS-column (Fig. 3A, Table 1D). Although the time spent in each CS column did not significantly differ for the unpaired group, the performance of these fish significantly varied across training trials (Fig. 3D, Table ...
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... of the paired group spent significantly more time in the CS+ column than in the CS-during both memory test 1 (Fig. 3B, Table 1E) and test 2 (Fig. 3C, Table 1F). Similarly to the paired group, the performance of unpaired fish did not significantly change between the two memory tests (Test effect: Χ 2 = 0.05, df = 1, p = Fig. 2 Simple conditioning. A Time (in seconds, median and quartiles) spent by fish from paired group (n = 20, grey) and unpaired ...
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... of the paired group spent significantly more time in the CS+ column than in the CS-during both memory test 1 (Fig. 3B, Table 1E) and test 2 (Fig. 3C, Table 1F). Similarly to the paired group, the performance of unpaired fish did not significantly change between the two memory tests (Test effect: Χ 2 = 0.05, df = 1, p = Fig. 2 Simple conditioning. A Time (in seconds, median and quartiles) spent by fish from paired group (n = 20, grey) and unpaired group (n = 18, white) in the ...
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... during simple classical associative (CS-US) conditioning ( Fig. 2A). B Linear model results for the performance of fish during memory test (Fig. 2B). C Linear model results for the performance of fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model ...
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... performance of fish during memory test (Fig. 2B). C Linear model results for the performance of fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model ...
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... fish during "generalization" test (Fig. 2C). D Linear mixed model results for the performance of fish (paired group) during discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant ...
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... discrimination conditioning (Fig. 3A). E Linear model results for the performance of fish (paired group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the ...
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... group) during memory test 1 (Fig. 3B). F Linear model results for the performance of fish (paired group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 45 s preceding the food reward release during the first ...
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... group) during memory test 2 (Fig. 3C). G Linear mixed model results for the performance of fish (unpaired group) during discrimination conditioning (Fig. 3D). E Linear model results for the performance of fish (unpaired group) during memory test 1 (Fig. 3E). F Linear model results for the performance of fish (unpaired group) during memory test 2 (Fig. 3F). Significant results are highlighted in green A Time (in seconds, median and quartiles) spent by fish from paired group (n = 18) in the CS+ (grey) and CS-(white) columns during the 45 s preceding the food reward release during the first conditioning trial of each conditioning day. B Time (in seconds, median and quartiles) spent by ...
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... group. *p < 0.05; **p < 0.01, ***p < 0.001; NS non-significant 0.81; Test:CS effect: F = 2.4, df = 1, p = 0.12). However, when analysing each test independently, fish from the unpaired group spent significantly more time in the CS1 (green) column than in the CS2 (red) one during test 1 (Fig. 3E, Table 1H), but this difference was absent in test 2 (Fig. 3F, Table ...
Citations
... Studying cognitive functions and learning in animals can be timeconsuming and labor-intensive. Automation may be advantageous in such studies as it can shorten the time needed for training and data acquisition (Buatois et al., 2024). In addition, automation can reduce unintended cueing by the experimenter. ...
... Automation has been successfully applied across various fish species and learning paradigms (Barreiros et al., 2021;Behrend et al., 1968;McKay et al., 2022;Pylatiuk et al., 2019;Singh et al., 2022). A recent study (Buatois et al., 2024) found a significant improvement in automated associative learning of zebrafish compared to the classical manual protocol (Colwill et al., 2005). We are only aware of two studies that directly compared the performance of fish in automated and manual training (Gatto et al., 2020;Gatto et al., 2021) and these reported poorer learning performance for some of the automated tasks. ...
... In contrast to simple microcontrollers, the Raspberry Zero W possesses a wireless connectivity module (Upton & Halfacree, 2014;Upton & Halfacree, 2016), which we used to create a local hotspot to operate the system remotely. This is a valuable addition to previous designs (Aoki et al., 2015;Buatois et al., 2024;Doyle et al., 2017;Jung et al., 2019) because it allows for an independent, remote communication with the system, making it also suitable for fieldwork (Häderer & Michiels, 2024;Henninger et al., 2018). This paper presents four applications featuring increasing levels of automation, ranging from simple pushbutton control to remote control and closed-loop procedures ( Figure 1). ...
Automation of experimental setups is a promising direction in behavioral research because it can facilitate the acquisition of data while increasing its repeatability and reliability. For example, research in spatial cognition can benefit from automated control by systematic manipulation of sensory cues and more efficient execution of training procedures. However, commercial solutions are often costly, restricted to specific platforms, and mainly focused on the automation of data acquisition, stimulus presentation, and reward delivery. Animal welfare considerations as well as experimental demands may require automating the access of an animal or animals to the experimental arena. Here, we provide and test a low‐cost, versatile Raspberry Pi‐based solution for such use cases. We provide four application scenarios of varying complexities, based on our research of spatial orientation and navigation in weakly electric fish, with step‐by‐step protocols for the control of gates in the experimental setups. This easy‐to‐implement, platform‐independent approach can be adapted to various experimental needs, including closed‐loop as well as field experiments. As such, it can contribute to the optimization and standardization of experiments in a variety of species, thereby enhancing the comparability of data.
... The zebrafish is an essential and crucial model organism in many fields of biomedical research including genetics, neurobehavior and pharmacology Rinkwitz et al., 2011;Buatois et al., 2023;Shishis et al., 2023). In many of these studies and procedures where a particular phenotype is to be measured, zebrafish are handled and removed from their home tank into a separate recording chamber or test tank by an external human experimenter (Shishis et al., 2023). ...
... Even in routine husbandry practices, zebrafish are often exposed to human handling which often include net chasing and removal from water into air (Pavlidis et al., 2013;Shishis et al., 2023). This can be highly distressing for the zebrafish as they are subjected to temporary hypoxia during transfer and possible aversive encounters with the external human experimenter if not handled correctly (Buatois et al., 2023;Pavlidis et al., 2013;Shishis et al., 2023). ...
... Zebrafish may be particularly sensitive to human handling, especially in finer-tuned behavioural studies where human handling may have acute, immediate, or short time-scale effects on results (Buatois et al., 2023;Shishis et al., 2023). It may also have chronic effects, if repeated handling is required, as is the case, e.g., for most learning studies conducted with the zebrafish (Ramsay et al., 2009;Gerlai, 2020c;Neely et al., 2018). ...
The zebrafish has been employed in several fields of biology due to its translational relevance and its simplicity and ease of
maintenance. Current industry standards favor keeping the largest
possible number of fish in the smallest possible volume of water to increase efficiency and reduce costs. However, physiological, and psychological stress resulting from such crowding may impact
a variety of phenotypes. Surprisingly, little is known about what constitutes an optimal housing environment for the zebrafish despite recent sporadic reports implying negative effects of the
standard practice of crowding. The purpose of this thesis was to investigate how housing condition dependent changes (tank size, housing density and handling) exert significant effects on the
zebrafish. By assessing zebrafish behaviour via a home tank observational procedure, the novel tank test, physiological whole-body cortisol analysis, water parameter analysis and handling
experiment, we hope to achieve a greater understanding of
stress/anxiety-inducing housing conditions in zebrafish.
Aquarium fish keeping is an incredibly popular hobby. Despite this popularity, fishes can suffer poor welfare due to being housed in an inappropriate environment, lack of owner knowledge that perpetuates misconceptions, and our perception of them as “lower vertebrates”. This article examines the complexity of fish biology and behaviour to support appropriate care of fishes within home aquaria. It focuses on the importance of evidence for what fish need and how to use such evidence in the domestic aquarium. In the UK, around 21% of households maintain an indoor aquarium and 13% of households have an outdoor pond. This equates to many millions of individual fish in private households. Approximately 70% of fishes in home aquaria are tropical freshwater species. Although fishes may appear easy pets to keep, being cheap to buy and readily available in different outlets, many common-in-the-home-aquarium species have specific requirements (from their water chemistry, physical environment, and social grouping) that they require to thrive, and owners should be aware of their natural biology and wild ecology when setting up an aquarium and maintaining a social group. Inaccuracies and misrepresentations abound when non-specialists think about fishes; they have no memory, they only grow to the size of the tank they are provided with, they do not feel pain and therefore are disposable. Fishes have complex physiologies that enable them to live in an environment alien to us as terrestrial mammals. They also display a diverse array of behaviours that provide them with fitness benefits within their habitat. Alongside essential aquarium considerations (heating, filtration, water quality), aquarium fish owners need to provide a suitably enriched environment for the species being housed. This case study considers simple steps that owners can take to improve welfare, health, and longevity of aquarium fishes through better knowledge of their natural history, the provision of a more ecologically relevant environment, and the maintenance of correct social groupings.
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