Quantity as a Fish Views It: Behavior and Neurobiology

Abstract

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

Keywords: fish cognition; imaging; pallium; quantity discrimination; retina; tectum; visual system; zebrafish.

FIGURE 1

Stimuli and neural circuits associated with prey capture in zebrafish larvae. (A) Looming-evoked escape (–) behavior in zebrafish larvae (Temizer et al., 2015). (B) A schematic representation of moving small or large dots entering the visual field of zebrafish larvae. Larvae tend to approach (+) small dots and avoid (–) large dots (Barker and Baier, 2015). (C) A schematic representation of moving small or large objects in zebrafish larvae. Small objects elicit an approach (+) interaction and large objects an avoidance (–) interaction (Preuss et al., 2014). (D) Classification of small and large objects in retinotectal circuits of zebrafish larvae eliciting an appetitive or aversive behavior in zebrafish larvae. Retinal ganglion cells (RGCs) detecting small objects project to the external layers of the zebrafish optic tectum. On the contrary, RGCs for large objects project to the deeper layer of the optic tectum (Preuss et al., 2014).

FIGURE 2

Stimuli and neural circuits involved in social behaviors. (A,B) Left: Several types of stimuli were presented to the visual field of zebrafish using a virtual reality assay. Black dots vary in angular size, vertical dimension, horizontal dimension, and number. At 7 dpf, these stimuli elicited an aversive turn with an increasing probability of repulsion with an increase in the retina occupancy. At 21 dpf, stimuli of the same size were considered attractive. Right: the model proposed by Harpaz et al. (2021) hypothesized the existence of two different populations in downstream areas that are responsible for attraction or avoidance of social stimuli. While only the repulsive population is mature at 7 dpf, the balance of excitation and inhibition of the two populations determines the behavior of the fish at 14–21 dpf. (C) Left: black dots with bout-like motion elicited shoaling behavior in juvenile zebrafish, while continuously moving dots were considered not attractive (Larsch and Baier, 2018). Right: Connectivity patterns of dorsal thalamus where Kappel et al. (2021) found a cluster of neurons selective for bout swim. Rth, rostral thalamus; POA, preoptic area; CMid, caudal midbrain; DT, dorsal thalamus; OT, optic tectum; Hyp, hypothalamus.

FIGURE 3

Schematic representation of the habituation/dishabituation paradigm used to identify neural correlates associated with a change in discrete and continuous magnitude in the adult zebrafish brain. (A) A change in number from 3 (habituation) to 9 (dishabituation) dots elicits an approach interaction. On the contrary, avoidance of the stimulus is detected in the change from 9 to 3 dots. An evaluation of immediate early genes (IEG) expressions revealed the main role of the thalamus and the dorso-central pallium (Dc) in the elaboration of changes in discrete quantity (numerosity) (Messina et al., 2020, 2022). (B) A change in size, i.e., a decrease (1/3x) or an increase (3x), revealed the main role of the retina and the optic tectum in the elaboration of continuous quantity (stimulus size) (Messina et al., 2020, 2022).

FIGURE 4

Whole-brain functional imaging to find neural substrate of zebrafish numerosity capability. (A) Schematic setup of the 2p-SPIM setup that allows calcium imaging of awake zebrafish larvae that are subjected to visual numerosity stimuli. (B) Examples of the numerosity stimuli, which consist of a particular number of black dots on a diffuse red background. The visual patterns are controlled for various non-numerosity continuous variables to find the responses that are intrinsic to the recognition of discrete numbers. (C) Schematic drawing of the brain. The forebrain includes the pallium (Pa) and the habenula (Hb). The midbrain contains the optic tectum (OT). (D) Representative results from 8-dpf larvae show neurons that exhibited different levels of activity during different stimuli (e.g., 2 versus 5 dots, etc.). Green: nuclei of identified neurons. Magenta: anatomical background generated via the maximum-intensity projection in Z of the standard deviation projection in time, depicting neurons with time-varying activity. The raw dataset covers the volume of ∼350 μm × 600 μm × 250 μm (depth), taken with three sections per volume for 42 min. (E) Representative activity traces, of the two neurons selected by arrowheads in panel (D), during number stimuli. The line graph depicts activity levels as ΔF/F for the time trial as shown, averaged over n = 50 trials. Neuron 1 (blue arrowhead): higher activity levels during the 5-dot stimuli compared to the 2-dot stimuli. Neuron 2 (yellow arrowhead): higher activity levels during the 2-dot stimuli compared to the 5-dot stimuli. P < 0.05, bootstrapping with resampling. Error bars represent SEM, n = 50. Scale bar in panel (D),100 μm.