Animals of many species are capable of "small data" learning, that is, of learning without repetition. Here we introduce larval Drosophila melanogaster as a relatively simple study case for such one-trial learning. Using odor-food associative conditioning, we first show that a sugar that is both sweet and nutritious (fructose) and sugars that are only sweet (arabinose) or only nutritious (sorbitol) all support appetitive one-trial learning. The same is the case for the optogenetic activation of a subset of dopaminergic neurons innervating the mushroom body, the memory center of the insects. In contrast, no onetrial learning is observed for an amino acid reward (aspartic acid). As regards the aversive domain, one-trial learning is demonstrated for high-concentration sodium chloride, but is not observed for a bitter tastant (quinine). Second, we provide follow-up, parametric analyses of odor-fructose learning. Specifically, we ascertain its dependency on the number and duration of training trials, the requirements for the behavioral expression of one-trial odor-fructose memory, its temporal stability, and the feasibility of one-trial differential conditioning. Our results set the stage for a neurogenetic analysis of one-trial learning and define the requirements for modeling mnemonic processes in the larva.

An adaptive transition from exploring the environment in search of vital resources to exploiting these resources once the search was successful is important to all animals. Here we study the neuronal circuitry that allows larval Drosophila melanogaster of either sex to negotiate this exploration-exploitation transition. We do so by combining Pavlovian conditioning with high-resolution behavioral tracking, optogenetic manipulation of individually identified neurons, and EM data-based analyses of synaptic organization. We find that optogenetic activation of the dopaminergic neuron DAN-i1 can both establish memory during training and acutely terminate learned search behavior in a subsequent recall test. Its activation leaves innate behavior unaffected, however. Specifically, DAN-i1 activation can establish associative memories of opposite valence after paired and unpaired training with odor, and its activation during the recall test can terminate the search behavior resulting from either of these memories. Our results further suggest that in its behavioral significance DAN-i1 activation resembles, but does not equal, sugar reward. Dendrogram analyses of all the synaptic connections between DAN-i1 and its two main targets, the Kenyon cells and the mushroom body output neuron MBON-i1, further suggest that the DAN-i1 signals during training and during the recall test could be delivered to the Kenyon cells and to MBON-i1, respectively, within previously unrecognized, locally confined branching structures. This would provide an elegant circuit motif to terminate search on its successful completion.

Dopamine serves many functions, and dopamine neurons are correspondingly diverse. We use a combination of optogenetics, behavioral experiments, and high-resolution videotracking to probe for the functional capacities of two single, identified dopamine neurons in larval Drosophila. The DAN-f1 and the DAN-d1 neuron were recently found to carry aversive teaching signals during Pavlovian olfactory learning. We enquire into a fundamental feature of these teaching signals, namely their temporal "fingerprint". That is, receiving punishment feels bad, whereas being relieved from it feels good, and animals and humans alike learn with opposite valence about the occurrence and the termination of punishment (the same principle applies in the appetitive domain, with opposite sign). We find that DAN-f1 but not DAN-d1 can mediate such timing-dependent valence reversal: presenting an odor before DAN-f1 activation leads to learned avoidance of the odor (punishment memory), whereas presenting the odor upon termination of DAN-f1 activation leads to learned approach (relief memory). In contrast, DAN-d1 confers punishment memory only. These effects are further characterized in terms of the impact of the duration of optogenetic activation, the temporal stability of the memories thus established, and the specific microbehavioral patterns of locomotion through which they are expressed. Together with recent findings in the appetitive domain and from adult Drosophila, our results suggest that heterogeneity in the temporal fingerprint of teaching signals might be a more general principle of reinforcement processing through dopamine neurons.

Adjusting behavior to changed environmental contingencies is critical for survival, and reversal learning provides an experimental handle on such cognitive flexibility. Here, we investigate reversal learning in larval Drosophila. Using odor-taste associations, we establish olfactory reversal learning in the appetitive and the aversive domain, using either fructose as a reward or high-concentration sodium chloride as a punishment, respectively. Reversal learning is demonstrated both in differential and in absolute conditioning, in either valence domain. In differential conditioning, the animals are first trained such that an odor A is paired, for example, with the reward whereas odor B is not (A+/B); this is followed by a second training phase with reversed contingencies (A/B+). In absolute conditioning, odor B is omitted, such that the animals are first trained with paired presentations of A and reward, followed by unpaired training in the second training phase. Our results reveal "true" reversal learning in that the opposite associative effects of both the first and the second training phase are detectable after reversed-contingency training. In what is a surprisingly quick, one-trial contingency adjustment in the Drosophila larva, the present study establishes a simple and genetically easy accessible study case of cognitive flexibility.

Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, the dopamine system has largely been studied using a rich genetic toolbox. Here, we suggest a complementary pharmacological approach applying the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling. Using Pavlovian conditioning, across developmental stages (3rd instar larva versus adult), valence domains (reward versus punishment), and types of reinforcement (natural versus optogenetically induced), we find that 3IY feeding specifically impairs associative learning, whereas additional feeding of L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine, rescues this impairment. This study establishes a simple, quick, and comparably low-cost approach that can be combined with the available genetic tools to manipulate and clarify the functions of the dopaminergic system - in D. melanogaster and other animals.