The Drosophila Larva as a Model for Studying Chemosensation and Chemosensory Learning: A Review

Understanding the relationship between brain and behavior is the fundamental challenge in neuroscience. We focus on chemosensation and chemosensory learning in larval Drosophila and review what is known about its molecular and cellular bases. Detailed analyses suggest that the larval olfactory system, albeit much reduced in cell number, shares the basic architecture, both in terms of receptor gene expression and neuronal circuitry, of its adult counterpart as well as of mammals. With respect to the gustatory system, less is known in particular with respect to processing of gustatory information in the central nervous system, leaving generalizations premature. On the behavioral level, a learning paradigm for the association of odors with food reinforcement has been introduced. Capitalizing on the knowledge of the chemosensory pathways, we review the first steps to reveal the genetic and cellular bases of olfactory learning in larval Drosophila. We argue that the simplicity of the larval chemosensory system, combined with the experimental accessibility of Drosophila on the genetic, electrophysiological, cellular, and behavioral level, makes this system suitable for an integrated understanding of chemosensation and chemosensory learnin

Associative Learning of Stimuli Paired and Unpaired With Reinforcement: Evaluating Evidence From Maggots, Flies, Bees, and Rats

Finding rewards and avoiding punishments are powerful goals of behavior. To maximize reward and minimize punishment, it is beneficial to learn about the stimuli that predict their occurrence, and decades of research have provided insight into the brain processes underlying such associative reinforcement learning. In addition, it is well known in experimental psychology, yet often unacknowledged in neighboring scientific disciplines, that subjects also learn about the stimuli that predict the absence of reinforcement. Here we evaluate evidence for both these learning processes. We focus on two study cases that both provide a baseline level of behavior against which the effects of associative learning can be assessed. Firstly, we report pertinent evidence from Drosophila larvae. A re-analysis of the literature reveals that through paired presentations of an odor A and a sugar reward (A+) the animals learn that the reward can be found where the odor is, and therefore show an above-baseline preference for the odor. In contrast, through unpaired training (A/+) the animals learn that the reward can be found precisely where the odor is not, and accordingly these larvae show a below-baseline preference for it (the same is the case, with inverted signs, for learning through taste punishment). In addition, we present previously unpublished data demonstrating that also during a two-odor, differential conditioning protocol (A+/B) both these learning processes take place in larvae, i.e., learning about both the rewarded stimulus A and the non-rewarded stimulus B (again, this is likewise the case for differential conditioning with taste punishment). Secondly, after briefly discussing published evidence from adult Drosophila, honeybees, and rats, we report an unpublished data set showing that relative to baseline behavior after truly random presentations of a visual stimulus A and punishment, rats exhibit memories of opposite valence upon paired and unpaired training. Collectively, the evidence conforms to classical findings in experimental psychology and suggests that across species animals associatively learn both through paired and through unpaired presentations of stimuli with reinforcement – with opposite valence. While the brain mechanisms of unpaired learning for the most part still need to be uncovered, the immediate implication is that using unpaired procedures as a mnemonically neutral control for associative reinforcement learning may be leading analyses astray

Behavioral Analyses of Sugar Processing in Choice, Feeding, and Learning in Larval Drosophila

Gustatory stimuli have at least 2 kinds of function: They can support immediate, reflexive responses (such as substrate choice and feeding) and they can drive internal reinforcement. We provide behavioral analyses of these functions with respect to sweet taste in larval Drosophila. The idea is to use the dose–effect characteristics as behavioral “fingerprints” to dissociate reflexive and reinforcing functions. For glucose and trehalose, we uncover relatively weak preference. In contrast, for fructose and sucrose, preference responses are strong and the effects on feeding pronounced. Specifically, larvae are attracted to, and feeding is stimulated most strongly for, intermediate concentrations of either sugar: Using very high concentrations (4 M) results in weakened preference and suppression of feeding. In contrast to such an optimum function regarding choice and feeding, an asymptotic dose–effect function is found for reinforcement learning: Learning scores reach asymptote at 2 M and remain stable for a 4-M concentration. A similar parametric discrepancy between the reflexive (choice and feeding) and reinforcing function is also seen for sodium chloride (Niewalda T, Singhal S, Fiala A, Saumweber T, Wegener S, Gerber B, in preparation). We discuss whether these discrepancies are based either on inhibition from high-osmolarity sensors upon specifically the reflexive pathways or whether different sensory pathways, with different effective dose–response characteristics, may have preferential access to drive either reflex responses or modulatory neurons mediating internal reinforcement, respectively

No evidence for visual context-dependency of olfactory learning in Drosophila

How is behaviour organised across sensory modalities? Specifically, we ask concerning the fruit fly Drosophila melanogaster how visual context affects olfactory learning and recall and whether information about visual context is getting integrated into olfactory memory. We find that changing visual context between training and test does not deteriorate olfactory memory scores, suggesting that these olfactory memories can drive behaviour despite a mismatch of visual context between training and test. Rather, both the establishment and the recall of olfactory memory are generally facilitated by light. In a follow-up experiment, we find no evidence for learning about combinations of odours and visual context as predictors for reinforcement even after explicit training in a so-called biconditional discrimination task. Thus, a ‘true’ interaction between visual and olfactory modalities is not evident; instead, light seems to influence olfactory learning and recall unspecifically, for example by altering motor activity, alertness or olfactory acuity

A rift between implicit and explicit conditioned valence in human pain relief learning

Pain is aversive, but does the cessation of pain (‘relief’) have a reward-like effect? Indeed, fruitflies avoid an odour previously presented before a painful event, but approach an odour previously presented after a painful event. Thus, event-timing may turn punishment to reward. However, is event-timing also crucial in humans who can have explicit cognitions about associations? Here, we show that stimuli associated with pain-relief acquire positive implicit valence but are explicitly rated as aversive. Specifically, the startle response, an evolutionarily conserved defence reflex, is attenuated by stimuli that had previously followed a painful event, indicating implicit positive valence of the conditioned stimulus; nevertheless, participants explicitly evaluate these stimuli as ‘emotionally negative’. These results demonstrate a rift between the implicit and explicit conditioned valence induced by pain relief. They might explain why humans in some cases are attracted by conditioned stimuli despite explicitly judging them as negative

High-resolution analysis of individual Drosophila melanogaster larvae uncovers individual variability in locomotion and its neurogenetic modulation

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its simple brain and genetic accessibility, the larva of the fruit fly Drosophila melanogaster allows one to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual, most studies of locomotion in larvae use measurements aggregated across animals, or animals tested one by one, an extravagance for larger-scale analyses. This prevents grasping the inter- and intra-individual variability in locomotion and its neurogenetic determinants. Here, we present the IMBA (individual maggot behaviour analyser) for analysing the behaviour of individual larvae within groups, reliably resolving individual identity across collisions. We use the IMBA to systematically describe the inter- and intra-individual variability in locomotion of wild-type animals, and how the variability is reduced by associative learning. We then report a novel locomotion phenotype of an adhesion GPCR mutant. We further investigated the modulation of locomotion across repeated activations of dopamine neurons in individual animals, and the transient backward locomotion induced by brief optogenetic activation of the brain-descending ‘mooncrawler’ neurons. In summary, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and its variability of individual larvae, with utility in multiple biomedical research contexts

Innate Attractiveness and Associative Learnability of Odors Can Be Dissociated in Larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X -- Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis

Four individually identified paired dopamine neurons signal reward in larval Drosophila

Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12]. It is thus warranted to understand which dopaminergic neurons mediate which function. We study larval Drosophila, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate- limiting enzyme of dopamine biosynthesis [ 5 and 13]. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order “cortical” brain region [ 1, 2, 3, 4, 5, 11, 12, 14 and 15]. We discover four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal [5], suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals [ 1, 2, 3, 4, 7, 9 and 10] (but see [6]). The numerical simplicity and connectomic tractability of the larval nervous system [ 16, 17, 18 and 19] now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior