Abstract Animals can adapt their behavioral responses to environmental cues by learning from experience. This ability relies on the formation and recall of memories that are shaped by beneficial or detrimental consequences and regulated by the dopaminergic system, which is highly conserved across insect species. In the Drosophila melanogaster larva, eight of total ∼120 dopaminergic neurons (DANs) innervate the mushroom body (MB), a key center for associative memory. This subset of DANs can be anatomically grouped into two clusters of four cells: the primary protocerebral anterior medial (pPAM) cluster, associated with reward signaling, and the dorsolateral 1 (DL1) cluster, associated with punishment. Such a functional dichotomy is observed in larval and adult Drosophila and reflects a fundamental organizational principle of reinforcement learning across invertebrate and even vertebrate species. Aversive reinforcement through high-salt exposure is encoded within the DL1 cluster in a combinatorial and heterogeneous manner, critically involving two neurons, DAN-f1 and DAN-g1. Using temporally precise optogenetic activation and inhibition during olfactory conditioning, we show that these neurons can modulate memory strength and valence. Their effects are most often consistent and predictable, enabling accurate computational modeling of DAN-driven teaching signals. By manipulating the intrinsic physiology of DAN-f1 and DAN-g1 and altering the valence of gustatory input, we are beginning to understand at the single-cell level how dopaminergic activity is systematically adjusted to control memory formation.

Dopaminergic neurons (DANs) play key roles in processing rewards and punishments across species. They evaluate sensory input, store memories, and update them based on relevance. To understand how individual DANs contribute to these functions, we studied Drosophila larvae, which have only about 120 DANs. Only eight of these project to the mushroom body (MB), a center for olfactory learning. These eight are divided into the pPAM and DL1 clusters, with four DANs each. We confirmed that pPAM neurons in the MB medial lobe encode sugar rewards. In the DL1 cluster, four neurons—DAN-c1, DAN-d1, DAN-f1, and DAN-g1—each target different MB regions. Notably, optogenetic activation of DAN-f1 and DAN-g1 can substitute for punishment. Additional methods (inhibition, calcium imaging, connectomics) show each DL1 DAN encodes a unique aspect of punishment, with DAN-g1 being pivotal for salt-based signals. Our findings reveal a clear division of labor among larval DL1 DANs for encoding punishment. The striking resemblance in the organizing principle of larval DANs with that of its adult counterpart and the mammalian basal ganglion suggests that there may be a limited number of efficient neural circuit solutions available to address more complex cognitive challenges in nature.

Drosophila larvae are able to associate an odor stimulus with a temporally overlapping teaching signal encoding reward or punishment. Here, we describe a standardized experimental setup that allows the analysis of larval aversive-odor-taste learning and memory. This is a Pavlovian learning experiment with a single training trial in which larvae are presented with two specific odors in succession, one odor together with salt at a high concentration that is harmful to the larva. In the subsequent test, the trained larvae then show avoidance of the salt-paired odor and spend more time near the unpaired odor. To rule out nonassociative effects (such as naive preferences for odors, exposure, or handling effects), two independent groups of larvae are reciprocally trained. Subsequently, the average of the two individual preference values is determined and quantified as a Performance Index (PI), which assigns a numerical value to the larvae's shown behavioral change.

Larvae of the fruit fly Drosophila melanogaster are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca2+ imaging, and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naive olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.SIGNIFICANCE STATEMENT The single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larval Drosophila as a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

The Drosophila larva has become an attractive model system for studying fundamental questions in neuroscience. Although the focus was initially on topics such as the structure of genes, mechanisms of inheritance, genetic regulation of development, and the function and physiology of ion channels, today it is often on the cellular and molecular principles of naive and learned behavior. Drosophila larvae have developed different mechanisms, often widespread in similar manifestations in the animal kingdom, to orient themselves toward olfactory, gustatory, mechanosensory, thermal, and visual stimuli to coordinate their locomotion appropriately. To adapt to changes in the environment, larvae are able to learn to categorize some of these sensory impressions as "good" or "bad." Depending on their relevance and reliability, the larva learns them and constantly updates these memories. Laboratory experiments allow us to parametrically study and describe many of these processes (e.g., olfactory appetitive and aversive memory or visual appetitive and aversive memory). Combining behavioral tests with various neurogenetic techniques allows us to thermally or optogenetically activate or inhibit individual cells during learning, memory consolidation, and memory retrieval. The molecular and genetic bases of larval learning can be analyzed by using specific mutants. The CRISPR-Cas method has established extensive new directions in this area, in addition to the already wide-ranging traditional approaches, like the GAL4/UAS system. The combination of these genetic methods with the simplicity and cost-effectiveness of the introduced behavioral assay provides a platform for discovering the fundamental mechanisms underlying learning and memory formation in the rather simple larval brain.

Drosophila larvae are able to associate an odor stimulus with a temporally overlapping teaching signal encoding reward or punishment. Here, we describe a standardized experimental setup that allows the analysis of larval aversive-odor-taste learning and memory. This is a Pavlovian learning experiment with a single training trial in which larvae are presented with two specific odors in succession, one odor together with salt at a high concentration that is harmful to the larva. In the subsequent test, the trained larvae then show avoidance of the salt-paired odor and spend more time near the unpaired odor. To rule out nonassociative effects (such as naive preferences for odors, exposure, or handling effects), two independent groups of larvae are reciprocally trained. Subsequently, the average of the two individual preference values is determined and quantified as a Performance Index (PI), which assigns a numerical value to the larvae's shown behavioral change.

TheDrosophilalarva has become an attractive model system for studying fundamental questions in neuroscience. Although the focus was initially on topics such as the structure of genes, mechanisms of inheritance, genetic regulation of development, and the function and physiology of ion channels, today it is often on the cellular and molecular principles of naive and learned behavior.Drosophilalarvae have developed different mechanisms, often widespread in similar manifestations in the animal kingdom, to orient themselves toward olfactory, gustatory, mechanosensory, thermal, and visual stimuli to coordinate their locomotion appropriately. To adapt to changes in the environment, larvae are able to learn to categorize some of these sensory impressions as “good” or “bad.” Depending on their relevance and reliability, the larva learns them and constantly updates these memories. Laboratory experiments allow us to parametrically study and describe many of these processes (e.g., olfactory appetitive and aversive memory or visual appetitive and aversive memory). Combining behavioral tests with various neurogenetic techniques allows us to thermally or optogenetically activate or inhibit individual cells during learning, memory consolidation, and memory retrieval. The molecular and genetic bases of larval learning can be analyzed by using specific mutants. The CRISPR–Cas method has established extensive new directions in this area, in addition to the already wide-ranging traditional approaches, like theGAL4/UASsystem. The combination of these genetic methods with the simplicity and cost-effectiveness of the introduced behavioral assay provides a platform for discovering the fundamental mechanisms underlying learning and memory formation in the rather simple larval brain.

How different sensory stimuli are collected, processed, and further transformed into a coordinated motor response is a fundamental question in neuroscience. In particular, the internal and external conditions that drive animals to switch to backward walking and the mechanisms by which the nervous system supports such behavior are still unknown. In fruit flies, moonwalker descending neurons (MDNs) are considered command-type neurons for backward locomotion as they receive visual and mechanosensory inputs and transmit motor-related signals to downstream neurons to elicit backward locomotion. Whether other modalities converge onto MDNs, which central brain neurons activate MDNs, and whether other retreat-driving pathways exist is currently unknown. Here, we show that olfactory stimulation can elicit MDN-mediated backward locomotion. Moreover, we identify the moonwalker subesophageal zone neurons (MooSEZs), a pair of bilateral neurons, which can trigger straight and rotational backward locomotion. MooSEZs act via postsynaptic MDNs and via other descending neurons. Although they respond to olfactory input, they are not required for odor-induced backward walking. Thus, this work reveals an important modality input to MDNs, a novel set of neurons presynaptic to MDNs driving backward locomotion and an MDN-independent backward locomotion pathway.

Abstract Although animals switch to backward walking upon sensing an obstacle or danger in their path, the initiation and execution of backward locomotion is poorly understood. The discovery of Moonwalker Descending Neurons (MDNs), made Drosophila useful to study neural circuits underlying backward locomotion. MDNs were demonstrated to receive visual and mechanosensory inputs. However, whether other modalities converge onto MDNs and what are the neural circuits activating MDNs are unknown. We show that aversive but not appetitive olfactory input triggers MDN-mediated backward locomotion. We identify in each hemisphere, a single Moonwalker Subesophageal Zone neuron (MooSEZ), which triggers backward locomotion. MooSEZs act both upstream and in parallel to MDNs. Surprisingly, MooSEZs also respond mostly to aversive odor. Contrary to MDNs, blocking MooSEZs activity has little effect on odor-evoked backward locomotion. Thus, this work reveals another important modality input to MDNs in addition to a novel olfactory pathway and MDN-independent backward locomotion pathway.

In many animals, the establishment and expression of food-related memory is limited by the presence of food and promoted by its absence, implying that this behavior is driven by motivation. In the past, this has already been demonstrated in various insects including honeybees and adult Drosophila. For Drosophila larvae, which are characterized by an immense growth and the resulting need for constant food intake, however, knowledge is rather limited. Accordingly, we have analyzed whether starvation modulates larval memory formation or expression after appetitive classical olfactory conditioning, in which an odor is associated with a sugar reward. We show that odor-sugar memory of starved larvae lasts longer than in fed larvae, although the initial performance is comparable. 80 minutes after odor fructose conditioning, only starved but not fed larvae show a reliable odor-fructose memory. This is likely due to a specific increase in the stability of anesthesia-resistant memory (ARM). Furthermore, we observe that starved larvae, in contrast to fed ones, prefer sugars that offer a nutritional benefit in addition to their sweetness. Taken together our work shows that Drosophila larvae adjust the expression of learned and naive choice behaviors in the absence of food. These effects are only short-lasting probably due to their lifestyle and their higher internal motivation to feed. In the future, the extensive use of established genetic tools will allow us to identify development-specific differences arising at the neuronal and molecular level.

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.