| Abstract: | Learning theories distinguish elemental from configural learning based on their different complexity. Although the former relies on simple and unambiguous links between the learned events, the latter deals with ambiguous discriminations in which conjunctive representations of events are learned as being different from their elements. In mammals, configural learning is mediated by brain areas that are either dispensable or partially involved in elemental learning. We studied whether the insect brain follows the same principles and addressed this question in the honey bee, the only insect in which configural learning has been demonstrated. We used a combination of conditioning protocols, disruption of neural activity, and optophysiological recording of olfactory circuits in the bee brain to determine whether mushroom bodies (MBs), brain structures that are essential for memory storage and retrieval, are equally necessary for configural and elemental olfactory learning. We show that bees with anesthetized MBs distinguish odors and learn elemental olfactory discriminations but not configural ones, such as positive and negative patterning. Inhibition of GABAergic signaling in the MB calyces, but not in the lobes, impairs patterning discrimination, thus suggesting a requirement of GABAergic feedback neurons from the lobes to the calyces for nonelemental learning. These results uncover a previously unidentified role for MBs besides memory storage and retrieval: namely, their implication in the acquisition of ambiguous discrimination problems. Thus, in insects as in mammals, specific brain regions are recruited when the ambiguity of learning tasks increases, a fact that reveals similarities in the neural processes underlying the elucidation of ambiguous tasks across species.Learning can be categorized into two levels of complexity termed elemental and configural (nonelemental) (1–3). Simple and unambiguous links between events characterize elemental learning (4). By contrast, ambiguity and nonlinearity characterize configural learning, where associations involve conjunctions of elemental stimuli, which may have different, contradictory outcomes. As a consequence, solving configural tasks typically requires treating stimulus conjunctions as being different from the simple sum of their elemental components (5–8). For example, in a negative patterning task (9–11), subjects have to discriminate a nonreinforced conjunction of two elements A and B from its reinforced elements (i.e., AB– vs. A+ and B+), which requires treating AB as being different from the simple sum of A and B (12, 13). The ambiguity of the task lies in the fact that each element (A and B) is as often reinforced (when presented alone) as nonreinforced (when presented as a compound). In mammals, different brain structures have been associated with these two learning forms: Whereas the hippocampus seems to be dispensable for learning elemental associations (6, 8), it is required for fast formation of conjunctive representations during learning tasks, such as spatial learning or contextual fear conditioning (6, 8, 10, 14–19). Moreover, the cortical system is necessary to form configural representations over extended training, thus supporting the learning of nonlinear discriminations,Here, we ask whether the specialization of different brain centers for learning tasks of different complexity is a property that can be extended to an insect brain. Insects offer the possibility of studying sophisticated behaviors and simultaneously accessing the neural bases of these behaviors (20). Several studies have shown that insects, in particular the honey bee Apis mellifera, possess higher-order cognitive abilities (5, 21), which raises the question of which neural mechanisms support these capacities in a brain whose size is only 1 mm3 (22).The mushroom bodies (MBs) are paired structures in the insect brain that have been historically associated with olfactory learning and memory. Their function has been extensively studied in a variety of elemental learning protocols, mainly in the honey bee and the fruit fly Drosophila melanogaster (23–29). In both species, MBs play a fundamental role for the encoding, storing, and retrieval of appetitive and aversive elemental memories, but no study has clearly established their role for nonelemental learning and memory (30). In fruit flies, this missing information may be due to the incapacity of these insects to solve nonelemental problems, such as negative patterning (31). By contrast, honey bees exhibit elaborated nonelemental learning abilities (32–36), which have been suggested to require intact MB function (5).Here, we used a combination of nonelemental conditioning protocols, disruption of MB function, and optophysiological recordings of neural activity to determine whether MBs are necessary for nonelemental forms of learning. Our results show that acquisition of olfactory patterning discriminations is impaired in bees in which neural activity in the MBs was blocked by procaine injection (37, 38), but not in control animals injected with saline solution. By contrast, MB blockade by procaine affected neither olfactory processing upstream of the MBs nor elemental olfactory discriminations. To uncover the neural mechanisms underlying the necessity of MBs for patterning discriminations, we focused on GABAergic feedback neurons (39), which provide inhibitory feedback to the MBs of the bee (40–43). We blocked GABAergic signaling by locally injecting picrotoxin (PTX), a GABA antagonist, into the MB calyces or into the MB lobes. We show that GABAergic feedback to the calyces—but not to the lobes—is required for patterning discriminations. These results uncover a previously unidentified role for MBs: namely, the disambiguation between elemental and conjunctive odor representations, thus supporting the learning of nonlinear discriminations. |
