A simple semi-automated home-tank method and procedure to explore classical associative learning in adult zebrafish.

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 zebrafish, including methods for the analysis of learning and memory in adult zebrafish. Perhaps the biggest obstacle in these methods is that zebrafish is particularly sensitive to human handling. To overcome this confound, automated learning paradigms have been developed with varying success. In this manuscript, we present a semi-automated home tank-based learning/memory test paradigm utilizing visual cues, and show that it is capable of quantifying classical associative learning performance in zebrafish. We demonstrate that in this task, zebrafish successfully acquire the association between coloured-light and food reward. The hardware and software components of the task are easy and cheap to obtain and simple to assemble and set up. The procedures of the paradigm allow the test fish to remain completely undisturbed by the experimenter for several days in their home (test) tank, eliminating human handling or human interference induced stress. We demonstrate that the development of cheap and simple automated home-tank-based learning paradigms for the zebrafish is feasible. We argue that such tasks will allow us to better characterize numerous cognitive and mnemonic features of the zebrafish, including elemental as well as configural learning and memory, which will, in turn, also enhance our ability to study neurobiological mechanisms underlying learning and memory using this model organism.

Acute administration of a dopamine D2/D3 receptor agonist alters behavioral and neural parameters in adult zebrafish.

The dopaminergic neurotransmitter system is implicated in several brain functions and behavioral processes. Alterations in it are associated with the pathogenesis of several human neurological disorders. Pharmacological agents that interact with the dopaminergic system allow the investigation of dopamine-mediated cellular and molecular responses and may elucidate the biological bases of such disorders. Zebrafish, a translationally relevant biomedical research organism, has been successfully employed in prior psychopharmacology studies. Here, we evaluated the effects of quinpirole (dopamine D2/D3 receptor agonist) in adult zebrafish on behavioral parameters, brain-derived neurotrophic factor (BDNF) and neurotransmitter levels. Zebrafish received intraperitoneal injections of 0.5, 1.0, or 2.0 mg/kg quinpirole or saline (control group) twice with an inter-injection interval of 48 h. All tests were performed 24 h after the second injection. After this acute quinpirole administration, zebrafish exhibited decreased locomotor activity, increased anxiety-like behaviors and memory impairment. However, quinpirole did not affect social and aggressive behavior. Quinpirole-treated fish exhibited stereotypic swimming, characterized by repetitive behavior followed by immobile episodes. Moreover, quinpirole treatment also decreased the number of BDNF-immunoreactive cells in the zebrafish brain. Analysis of neurotransmitter levels demonstrated a significant increase in glutamate and a decrease in serotonin, while no alterations were observed in dopamine. These findings demonstrate that dopaminergic signaling altered by quinpirole administration results in significant behavioral and neuroplastic changes in the central nervous system of zebrafish. Thus, we conclude that the use of quinpirole administration in adult zebrafish may be an appropriate tool for the analysis of mechanisms underlying neurological disorders related to the dopaminergic system.

The Neural Signature of Visual Learning Under Restrictive Virtual-Reality Conditions.

Honey bees are reputed for their remarkable visual learning and navigation capabilities. These capacities can be studied in virtual reality (VR) environments, which allow studying performances of tethered animals in stationary flight or walk under full control of the sensory environment. Here, we used a 2D VR setup in which a tethered bee walking stationary under restrictive closed-loop conditions learned to discriminate vertical rectangles differing in color and reinforcing outcome. Closed-loop conditions restricted stimulus control to lateral displacements. Consistently with prior VR analyses, bees learned to discriminate the trained stimuli. Ex vivo analyses on the brains of learners and non-learners showed that successful learning led to a downregulation of three immediate early genes in the main regions of the visual circuit, the optic lobes (OLs) and the calyces of the mushroom bodies (MBs). While Egr1 was downregulated in the OLs, Hr38 and kakusei were coincidently downregulated in the calyces of the MBs. Our work thus reveals that color discrimination learning induced a neural signature distributed along the sequential pathway of color processing that is consistent with an inhibitory trace. This trace may relate to the motor patterns required to solve the discrimination task, which are different from those underlying pathfinding in 3D VR scenarios allowing for navigation and exploratory learning and which lead to IEG upregulation.

Visual learning in a virtual reality environment upregulates immediate early gene expression in the mushroom bodies of honey bees.

Free-flying bees learn efficiently to solve numerous visual tasks. Yet, the neural underpinnings of this capacity remain unexplored. We used a 3D virtual reality (VR) environment to study visual learning and determine if it leads to changes in immediate early gene (IEG) expression in specific areas of the bee brain. We focused on kakusei, Hr38 and Egr1, three IEGs that have been related to bee foraging and orientation, and compared their relative expression in the calyces of the mushroom bodies, the optic lobes and the rest of the brain after color discrimination learning. Bees learned to discriminate virtual stimuli displaying different colors and retained the information learned. Successful learners exhibited Egr1 upregulation only in the calyces of the mushroom bodies, thus uncovering a privileged involvement of these brain regions in associative color learning and the usefulness of Egr1 as a marker of neural activity induced by this phenomenon.

Colored-Light Preference in Zebrafish (Danio rerio).

Over the past decade, the zebrafish has been increasingly employed in biomedical neuroscience research due to its numerous evolutionarily conserved features with mammals. Its simple brain and the several molecular tools available for this species make the zebrafish an appealing model to study mechanisms of complex brain functions, including learning and memory. Most learning paradigms developed for the zebrafish have employed visual stimuli as the associative cue. Spontaneous color preference is a potential confound in such studies. It has been analyzed in zebrafish using colored objects, but with conflicting results. It has rarely been explored with colored light, despite the increasing use of computer-generated visual stimuli. Here, we employ a light emitting diode (RGB-system) light-based color preference task in the plus-maze. In two independent experiments, zebrafish were tested in a four-choice or dual-choice condition by using four different-colored lights (red, green, blue and yellow). Our results suggest a light preference hierarchy that depends on context, since yellow was preferred over green in the four-choice condition whereas blue was preferred over all other colors in the two-choice condition. These results are useful for future color-light-based learning experiments in zebrafish.

Elemental and Configural Associative Learning in Spatial Tasks: Could Zebrafish be Used to Advance Our Knowledge?

Spatial learning and memory have been studied for several decades. Analyses of these processes pose fundamental scientific questions but are also relevant from a biomedical perspective. The cellular, synaptic and molecular mechanisms underlying spatial learning have been intensively investigated, yet the behavioral mechanisms/strategies in a spatial task still pose unanswered questions. Spatial learning relies upon configural information about cues in the environment. However, each of these cues can also independently form part of an elemental association with the specific spatial position, and thus spatial tasks may be solved using elemental (single CS and US association) learning. Here, we first briefly review what we know about configural learning from studies with rodents. Subsequently, we discuss the pros and cons of employing a relatively novel laboratory organism, the zebrafish in such studies, providing some examples of methods with which both elemental and configural learning may be explored with this species. Last, we speculate about future research directions focusing on how zebrafish may advance our knowledge. We argue that zebrafish strikes a reasonable compromise between system complexity and practical simplicity and that adding this species to the studies with laboratory rodents will allow us to gain a better understanding of both the evolution of and the mechanisms underlying spatial learning. We conclude that zebrafish research will enhance the translational relevance of our findings.

Higher-order discrimination learning by honeybees in a virtual environment.

Non-elemental learning constitutes a cognitive challenge because events to be learned are usually ambiguous in terms of reinforcement outcome, contrary to elemental learning, which relies on unambiguous associations. Negative patterning (NP) constitutes a paradigmatic case of non-elemental learning, as subjects have to learn that single elements are reinforced while their simultaneous presentation is not reinforced (A+, B+ vs. AB-). Solving NP requires treating AB as being different from the linear sum of its components in order to overcome the ambiguity of stimulus reinforcement (i.e. A and B are as often reinforced as not reinforced). The honeybee is currently the only insect mastering NP as shown by studies restricted mainly to the olfactory domain. Here, we tested the bees' capacity to solve a NP discrimination in the visual domain and used to this end a virtual reality (VR) environment in which a tethered bee walking stationary on a spherical treadmill faces visual stimuli projected on a semicircular screen. We show that bees learn a composite grating made of alternated green and blue bars in an elemental way, and generalize their response to both a blue and a green grating. Yet, after NP training, one-quarter of the bees inhibited elemental processing and responded significantly more to the single-coloured gratings than to the composite grating. Alternative strategies were used by the other bees, which achieved partial NP learning. These results offer attractive perspectives to study different forms of visual learning in a controlled VR environment, and dissect their underlying mechanisms.

Transfer of Visual Learning Between a Virtual and a Real Environment in Honey Bees: The Role of Active Vision.

To study visual learning in honey bees, we developed a virtual reality (VR) system in which the movements of a tethered bee walking stationary on a spherical treadmill update the visual panorama presented in front of it (closed-loop conditions), thus creating an experience of immersion within a virtual environment. In parallel, we developed a small Y-maze with interchangeable end-boxes, which allowed replacing repeatedly a freely walking bee into the starting point of the maze for repeated decision recording. Using conditioning and transfer experiments between the VR setup and the Y-maze, we studied the extent to which movement freedom and active vision are crucial for learning a simple color discrimination. Approximately 57% of the bees learned the visual discrimination in both conditions. Transfer from VR to the maze improved significantly the bees' performances: 75% of bees having chosen the CS+ continued doing so and 100% of bees having chosen the CS- reverted their choice in favor of the CS+. In contrast, no improvement was seen for these two groups of bees during the reciprocal transfer from the Y-maze to VR. In this case, bees exhibited inconsistent choices in the VR setup. The asymmetric transfer between contexts indicates that the information learned in each environment may be different despite the similar learning success. Moreover, it shows that reducing the possibility of active vision and movement freedom in the passage from the maze to the VR impairs the expression of visual learning while increasing them in the reciprocal transfer improves it. Our results underline the active nature of visual processing in bees and allow discussing the developments required for immersive VR experiences in insects.

Using virtual reality to study visual performances of honeybees.

Virtual reality (VR) offers an appealing experimental framework for studying visual performances of insects under highly controlled conditions. In the case of the honeybee Apis mellifera, this possibility may fill the gap between behavioural analyses in free-flight and cellular analyses in the laboratory. Using automated, computer-controlled systems, it is possible to generate virtual stimuli or even entire environments that can be modified to test hypotheses on bee visual behaviour. The bee itself can remain tethered in place, making it possible to record neural activity while the bees is performing behavioural tasks. Recent studies have examined visual navigation and attentional processes in VR on flying or walking tethered bees, but experimental paradigms for examining visual learning and memory are only just emerging. Behavioural performances of bees under current experimental conditions are often lower in VR than in natural environments, but further improvements on current experimental protocols seem possible. Here we discuss current developments and conclude that it is essential to tailor the specifications of the VR simulation to the visual processing of honeybees to improve the success of this research endeavour.

Associative visual learning by tethered bees in a controlled visual environment.

Free-flying honeybees exhibit remarkable cognitive capacities but the neural underpinnings of these capacities cannot be studied in flying insects. Conversely, immobilized bees are accessible to neurobiological investigation but display poor visual learning. To overcome this limitation, we aimed at establishing a controlled visual environment in which tethered bees walking on a spherical treadmill learn to discriminate visual stimuli video projected in front of them. Freely flying bees trained to walk into a miniature Y-maze displaying these stimuli in a dark environment learned the visual discrimination efficiently when one of them (CS+) was paired with sucrose and the other with quinine solution (CS-). Adapting this discrimination to the treadmill paradigm with a tethered, walking bee was successful as bees exhibited robust discrimination and preferred the CS+ to the CS- after training. As learning was better in the maze, movement freedom, active vision and behavioral context might be important for visual learning. The nature of the punishment associated with the CS- also affects learning as quinine and distilled water enhanced the proportion of learners. Thus, visual learning is amenable to a controlled environment in which tethered bees learn visual stimuli, a result that is important for future neurobiological studies in virtual reality.

Evidence of trapline foraging in honeybees.

Central-place foragers exploiting floral resources often use multi-destination routes (traplines) to maximise their foraging efficiency. Recent studies on bumblebees have showed how solitary foragers can learn traplines, minimising travel costs between multiple replenishing feeding locations. Here we demonstrate a similar routing strategy in the honeybee (Apis mellifera), a major pollinator known to recruit nestmates to discovered food resources. Individual honeybees trained to collect sucrose solution from four artificial flowers arranged within 10 m of the hive location developed repeatable visitation sequences both in the laboratory and in the field. A 10-fold increase of between-flower distances considerably intensified this routing behaviour, with bees establishing more stable and more efficient routes at larger spatial scales. In these advanced social insects, trapline foraging may complement cooperative foraging for exploiting food resources near the hive (where dance recruitment is not used) or when resources are not large enough to sustain multiple foragers at once.