Vertebrates and some invertebrates have developed neural systems that allow the association of non-familiar tastes with visceral toxic effects, resulting in a learned aversion (known as conditioned taste aversion or CTA) to tastes with similar taste characteristics [10]. The learned aversion results in an adaptative response of rejection to the toxic food that induced gastrointestinal malaise (thus reducing the probability of toxic experiences) and an acquired perception of unpleasant taste. This type of taste learning is useful for preserving life, particularly for omnivorous species, including human beings. The acquired taste aversion can be extinguished if further experiences with the same taste stimulus (or even similar taste stimuli) are not associated with poisoning or negative consequences.

CTA and CTA extinction can be reproduced in the laboratory with animal models in order to explore the neurobiological and behavioural characteristics of this learning [28,29]. In the laboratory (as in nature), CTA is characterised by the fact that taste aversion can be acquired through a single association between the taste stimulus and visceral malaise, and because the association between the novel taste and visceral consequences is possible even some hours after the processing of both stimuli [30,31]. This characteristic of CTA, to resist long delays between stimuli, is explained by the physiological processes of digestion, which implies a delay between the taste processing and visceral consequences. Considering this physiological time interval between the taste processing and the gastrointestinal malaise, CTA could result from the association between the taste memory’s trace and the visceral disease. The CTA paradigm used in the laboratory is a useful tool to investigate the biological and behavioural bases of aversive learning and taste memory, not only regarding the specificity of CTA but also with respect to other taste learning phenomena, such as neophobia (a precautionary response to the first exposure to a novel food or taste) or latent inhibition of CTA (a reduced conditioned aversion resulting from taste pre-exposures without negative or aversive consequences) [32,33,34,35]. An example of the relevance of the CTA paradigm is the finding that physical (external) and internal (such as the time of day) cues can separately modulate the magnitude of a learned aversion [36,37,38].

The conventional CTA paradigm includes ad libitum feeding throughout the procedure and a daily water restriction baseline stage to facilitate similar physiological states among the animals. The next stage is the conditioning session. In this stage, the animals are exposed to a novel taste stimulus (the conditioning stimulus, CS, in the terminology of associative learning). In the CTA paradigm, taste stimuli are usually provided as fluids (for example, as saccharin or sodium chloride solutions) to facilitate intake measurement. Around 20 min after intake, an intraperitoneal injection of lithium chloride (LiCl 0.15 M, 2% of body weight) is administrated to induce gastrointestinal malaise (the unconditioned stimulus, US). To a lesser extent, other aversive stimuli have also been used to induce taste aversion [39,40]. A recovery day with ad libitum water usually follows this stage. The magnitude of the taste aversion is evaluated the next day (testing day), by one-bottle (CS) or two-bottle (CS vs. water) tests, according to the amounts consumed. Figure 2 shows the CTA paradigm stages. This paradigm is used to explore the neurobiology of taste aversion learning and memory.

A complex nervous system mechanism is involved in the acquisition of CTA [41,42]. Animal models provide critical data to understand the neuroanatomy and neurobiology of taste learning and memory. The neural network of taste aversion learning and memory includes the nucleus of the solitary tract (NTS), the posteromedial pontine parabrachial nucleus, the lateral hypothalamus, the bed nucleus of the stria terminalis, the amygdala and the ventroposteromedial and lateral thalamus. The superior cortical level of processing has been described in the gustatory insular cortex region. The functional connectivity between the parabrachial nucleus and the gustatory insular cortex is selectively involved in the acquisition of CTA but not in the formation of safe taste memories [43]. These pathways and the vagal system involved in the processing of visceral malaise are necessary for the acquisition of CTA and taste aversion memory [10,11,12,14]. Moreover, the role of other brain structures in the neurobiology of CTA, such as the medial prefrontal cortex and the nucleus accumbens, is being elucidated at present [44], together with the functions of the piriform [45] and perirhinal [46] cortices in taste recognition. The neural system of CTA involves the activity of this brain and brainstem network, but the specific functions of each component are not fully understood. Although the CTA mechanisms of the gustatory insular cortex and parabrachial nucleus are well described, the involvement of the amygdala and its nuclei in specific processes of taste aversion learning and memory is not fully known [47,48,49]. Animal lesion studies have pointed to the central and basolateral nuclei of the amygdala as the amygdaloid nuclei with specific functions in taste aversion learning and memory [50]. However, the basolateral amygdala seems to be the main nucleus involved in the acquisition of CTA [49], probably modulating the magnitude of taste aversion [51,52]. A possible mechanism by which basolateral amygdala can modulate the intensity of CTA is through the neophobia phenomenon, considering that this nucleus is implicated in the perception of novelty of taste stimuli. The correct processing of novelty is one of the mechanisms affecting the magnitude of CTA [53]. In addition to lesion studies, other methods and approaches have also pointed to the basolateral amygdala as a selective amygdaloid nucleus mediating the acquisition of CTA. By two-photon calcium imaging it has been revealed that a CTA-dependent neuronal activation of specific neurons of the insular cortex that project to the basolateral amygdala [54], and chemical activation of the insular cortex-basolateral amygdala projection by Clozapine-N-oxide after taste exposure, can induce aversive taste memory in mice [55]. Thus, the function of the basolateral amygdala on CTA might be controlled by afferent axons from the gustatory insular cortex [53]. Moreover, molecular studies have supported the relevance of this cortico-amygdaloid projection for the formation of CTA [56,57]. It can be concluded that specific connections between the gustatory insular cortex and the basolateral complex of the amygdala [49,57,58], and between the amygdala and the brainstem nuclei involved in CTA [12,47,48], could be recruited to influence the intensity of acquired taste aversions.

Some molecular mechanisms are specific to certain forms of taste learning and memory [59]. However, learning of novel tastes, taste familiarity and taste aversion extinction share biological pathways and mechanisms with CTA. The transcriptional processes necessary for the acquisition of taste learning or processing of taste novelty that occur in the gustatory insular cortex have been described in rats [60]. Particularly, this model has provided relevant information about how novel taste experience, a process that strengthens the acquisition of CTA, modifies the genetic transcription in this cortical area during taste memory consolidation [60]. Likewise, taste learning of novel or familiar tastes promotes different changes in the transcriptome of this cortical region. Moreover, the consolidation of positive or negative taste learning (according to its positive or negative visceral consequences) also induces transcriptional activity in this region [60]. Learning of novel tastes induces biochemical alterations in the gustatory insular cortex of other rodents as well, including increased cholinergic activity [61], and changes in protein phosphorylation [62], facilitating taste memory consolidation [63,64]. Furthermore, several studies have shown that taste memory consolidation is altered after pharmacological inhibition of protein synthesis in the gustatory insular cortex [65,66]. Recently [67], the administration of protein synthesis inhibitors directly into the gustatory insular cortex during long-term taste memory acquisition altered the formation of long-term memory. However, this procedure did not affect memory persistence when these inhibitors were infused 3 days after the memory formation. Interestingly, the infusion of protein synthesis inhibitors 14 days after the memory acquisition increased the memory persistence [67], which suggests that long-term memory may be altered by protein synthesis inhibitors, even several days after the formation of taste memory.

Other molecular mechanisms of taste learning that could participate in CTA have been seen in the gustatory insular cortex. Various immediate early genes identified in this cortical region during taste learning, such as the activity-regulated cytoskeleton associated protein (Arc)/Arg3.1 gene, seem to regulate the excitability of synapses associated with synaptic plasticity processes and long-term taste memory [68,69]. The function of the Arc/Arg3.1 protein appears to be different depending on the specificity of taste learning, since novel taste learning can increase as well as reduce the expression of this protein in the gustatory insular cortex, according to specific time points. These transcriptional changes may last for hours and are more intense compared to the processing of familiar tastes [60]. Moreover, a hemispheric lateralisation of the expression of Arc/Arg3.1 protein is observed in the gustatory insular cortex related to the processing of novel tastes [70].

The neural plasticity mechanisms related to taste learning, including CTA, also involve other molecular sequences. The expression of brain-derived neurotrophic factor (BDNF) in the gustatory insular cortex (and the basolateral amygdala) induces long-term synaptic plasticity, and the acquisition of taste aversions seems to block the long-lasting BDNF-induced strengthening of synaptic plasticity [71]. These findings point to the BDNF gene expression in the gustatory insular cortex as one of the molecular mechanisms critically involved in the long-term synaptic plasticity processes related to taste memory. Additional gene expressions have been found in the gustatory insular cortex during taste learning, including the expression of c-fos [72], Homer1a [73] and the transcription factor Elk-1 [74]. The functions of these gene expressions and their respective proteins are unclear, although it is assumed that they are a relevant part of the synaptic plasticity necessary for taste learning and memory [75]. All this evidence indicates that the gene expression in the gustatory insular cortex and in other brain regions involved in taste learning is a molecular key for the acquisition of taste learning and the subsequent taste memory [63].