The interest in the study of emotion in non-human animals dates back to the publication of Darwin’s monograph “The Expression of Emotions in Man and Animals”1. However, the fact that human emotions are subjectively experienced as feelings has raised difficulties in defining emotion in animals in objective scientific terms2. Nevertheless, other dimensions of emotion, namely the expression of emotion-specific behaviour and accompanying physiological responses, have been documented in many species and a consensus has emerged that animals should express organismic states that index occurrences of value in the environment, regardless of whether these states are consciously experienced2,3,4,5. These organismic states would be triggered by the value, in terms of potential impact in Darwinian fitness, that an animal ascribes to a stimulus and they would instantiate the ability to respond adaptively to environmental threats (e.g. presence of predators; presence of competitors for resources, such as shelters or territories) and opportunities (e.g. mating partners; food availability; possibility of ascending in social hierarchy). Within this framework, these global organismic states, and their behavioural expression, represent the organism’s experience of reward and threat, and as such they can be seen as similar to human core affect states4. The evolution of core affect states (or central emotion states sensu 5) in animals is plausible as it would provide a way for animals to maximize the acquisition of fitness-enhancing rewards while, simultaneously, minimizing exposure to fitness-threatening punishers. Moreover, these emotion-like states are characterized by general functional properties (i.e. scalability, valence, persistence and generalization) that apply across species and thus make them recognizable and suitable for phylogenetic studies of emotion5. Recently, this approach has been used to describe, at the behavioural level, the occurrence of a core affect state of defensive arousal in fruit flies repeatedly exposed to a threat stimulus6. Thus, the stage has been set for documenting the occurrence of core affect states across phylogeny and to study how evolutionary conserved are the molecular mechanisms and neural circuits underlying them.

In human research core affect has been conceptualized as a dimensional characterization of the emotion experience along two fundamental underlying dimensions: valence (positive/negative) and intensity (or arousal)7,8. Hence, core affect can be represented in a two-dimensional space, which became known as the circumplex model of affect9, where these two variable define 4 quadrants (Q): Q1 = Positive affect, high arousal (e.g. happiness); Q2 = positive affect, low arousal (e.g. (relaxed mood); Q3 = negative affect, low arousal (e.g. sadness); Q4 = negative affect, high arousal (e.g. fear). The extension of this model to emotion-like states in animals has been proposed by4, who suggested that the axis Q3-Q1 defines a reward acquisition system, with Q1 representing appetitive motivational states that facilitate seeking and obtaining rewards and Q3 representing loss or lack of reward and associated low activity states, whereas the axis Q2-Q4 defines a punishment avoidance system, with Q4 associated to active responses to the presence of threat and Q2 to passive responses to low levels of threat. In humans, non-human primates and rodents, where the neural substrates of emotion have been more extensively studied, these two core affect axes have been associated with different neural mechanisms. Reward acquisition relies on the mesolimbic dopaminergic system, in particular the prefrontal cortex and specific hedonic hotspots located in the ventral striatum (e.g. nucleus accumbens)10,11, whereas punishment avoidance has been associated either with the fight-or-flight system (in Q4), or with the behavioural inhibition system (in Q2), with the amygdala playing a central role in either case2,4.

In order to create internal emotion-like states that support adaptive physiological and behavioural responses towards ecological threats or opportunities, animals must have evolved perceptual and cognitive mechanisms that identify reliable cues in the environment (i.e. aversive vs. appetitive stimuli, respectively)12. When specific environmental cues deterministically predict an appropriate response, these responses can be simple reflexes and fixed action patterns elicited by these cues. However, when environmental complexity and variability increase, single environmental cues may no longer be informative and the evolution of appraisal mechanisms that cognitively assess the presence of threats and opportunities in the environment is predicted13,14. According to cognitive theories of emotion, individuals continuously monitor the environment using a set of stimulus evaluation checks (e.g. intrinsic valence, novelty, prediction error, capacity for control) in order to evaluate the valence (positive/negative) and salience (high/low) of detected stimuli, and also assess the available organismal resources to deal with them (i.e. coping mechanisms)15,16. The outcome of appraisal translates into an adjustment of the core affective state of the animal to the perceived state of the external environment. Although an integrated study of the different stimulus evaluation checks used by animals is still lacking, empirical evidence for the occurrence of each of these checks has been described in a wide range of animals, from fish to mammals (see16 for a recent review).

In this study we have used the Gilthead Sea Bream (Sparus aurata) to study if perceived stimulus valence (i.e. appetitive vs. aversive) and salience (i.e. high vs. low) trigger specific behavioural, physiological and brain states, indicative of stimulus-appraisal driven emotion-like states in fish. We have selected this species given its economic importance in European aquaculture, which gives an added value to our results in terms of implications for the assessment of welfare of farmed fish17. We have used two stimuli with different intrinsic valences (appetitive: food; aversive: physical constraint) that were presented to the focal individuals in a predictable or unpredictable manner. Predictability was used as a proxy of stimulus salience. The effect of predictability as an appraisal modulator has already been documented in other fish species, both towards aversive and appetitive stimuli18. Thus, if emotion-like core affect states are also present in fish we predict that each of the four valence x predictability (salience) treatments will elicit specific brain and physiological states and behavioural profiles, which correspond to each of the four quadrants of the circumplex model of affect described above, namely: Q1 = unpredictable appetitive (UnPRDapp); Q2 = predictable appetitive (PRDapp); Q3 = predictable aversive (PRDavr); Q4 = unpredictable aversive (UnPRDavr).

Brain states for each treatment were characterized using the expression of a set of immediate early genes (see below), as markers of neural activity, in a set of brain regions homologous to those known to be involved in reward and aversion processing in mammals19, namely the medial zone of the dorsal telencephalic area (Dm, putative homologue of the mammalian basolateral amygdala); lateral zone of the dorsal telencephalic area (Dl, hippocampus homologue); ventral nucleus of the ventral telencephalic area (Vv, septum homologue)20,21. Immediate early genes are expressed in response to external stimuli without requiring previous protein synthesis and act as effector genes, changing the metabolism of the cell, or as transcription factors, orchestrating the cellular profile of gene expression. Consequently, in the field of neuroscience they have been widely used as markers of neuronal activity to map patterns of brain activation in response to specific stimuli or to behavioural tasks22. However, it is often the case that different immediate early genes provide different pictures of brain activation22, which is most probably due to fact that they are involved in multiple parallel signalling pathways. Thus, we have used the expression of four different immediate early genes [early growth response 1 (egr-1), FBJ osteosarcoma oncogene (c-fos), brain-derived neurotrophic factor (bdnf) and neuronal PAS domain protein 4a (npas4)] to characterize central brain states in fish exposed to the different treatments. We have studied the response of each of these genes independently of the others because it is possible that a specific signalling pathway is more related to emotional responses than others, but we have also studied the integrated response of the four genes as an overall neurogenomic state in response to emotional stimuli, as indicated by the patterns of gene co-expression in each brain region. Circulating cortisol levels were used as a marker of the activity of the hypothalamic-pituitary-interrenal axis, which is a major player in the integrated organismal response to environmental stimuli23. Finally, behavioural states were characterized by the expression of observed behavioural patterns, namely, social interactions and escape attempts.