Cognitive limits of larval Drosophila: testing for conditioned inhibition, sensory preconditioning, and second-order conditioning.

Drosophila larvae are an established model system for studying the mechanisms of innate and simple forms of learned behavior. They have about 10 times fewer neurons than adult flies, and it was the low total number of their neurons that allowed for an electron microscopic reconstruction of their brain at synaptic resolution. Regarding the mushroom body, a central brain structure for many forms of associative learning in insects, it turned out that more than half of the classes of synaptic connection had previously escaped attention. Understanding the function of these circuit motifs, subsequently confirmed in adult flies, is an important current research topic. In this context, we test larval Drosophila for their cognitive abilities in three tasks that are characteristically more complex than those previously studied. Our data provide evidence for (i) conditioned inhibition, as has previously been reported for adult flies and honeybees. Unlike what is described for adult flies and honeybees, however, our data do not provide evidence for (ii) sensory preconditioning or (iii) second-order conditioning in Drosophila larvae. We discuss the methodological features of our experiments as well as four specific aspects of the organization of the larval brain that may explain why these two forms of learning are observed in adult flies and honeybees, but not in larval Drosophila.

Prediction error drives associative learning and conditioned behavior in a spiking model of Drosophila larva.

Predicting reinforcement from sensory cues is beneficial for goal-directed behavior. In insect brains, underlying associations between cues and reinforcement, encoded by dopaminergic neurons, are formed in the mushroom body. We propose a spiking model of the Drosophila larva mushroom body. It includes a feedback motif conveying learned reinforcement expectation to dopaminergic neurons, which can compute prediction error as the difference between expected and present reinforcement. We demonstrate that this can serve as a driving force in learning. When combined with synaptic homeostasis, our model accounts for theoretically derived features of acquisition and loss of associations that depend on the intensity of the reinforcement and its temporal proximity to the cue. From modeling olfactory learning over the time course of behavioral experiments and simulating the locomotion of individual larvae toward or away from odor sources in a virtual environment, we conclude that learning driven by prediction errors can explain larval behavior.

Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila.

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.

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.

The making of a maggot brain.

The way neurons in the brain rewire in larvae as they turn to adult fruit flies sheds light on how complete metamorphosis was 'invented' over the course of evolution.

Age-related decrease in appetitive associative memory in fruit flies.

Memory scores are dynamic across developmental stages. In particular, memory scores typically decrease from late adolescence into old age, reflecting complex changes in mnemonic and sensory-motor faculties, metabolic and motivational changes, and changes in cognitive strategy as well. In Drosophila melanogaster, such age-related decreases in memory scores have been studied intensely for the association of odours with electric shock punishment. We report that odour-sucrose reward memory scores likewise decrease as the flies age. This was observed after one-trial and after two-trial conditioning, and for both immediate testing and recall tests 1 day later. This decrease was particularly pronounced in relatively young animals, in the first 2-3 weeks after adult hatching, and was more pronounced in female than in male flies.

Optogenetically induced reward and 'frustration' memory in larval Drosophila melanogaster.

Animals, including humans, form oppositely valenced memories for stimuli that predict the occurrence versus the termination of a reward: appetitive 'reward' memory for stimuli associated with the occurrence of a reward and aversive 'frustration' memory for stimuli that are associated with its termination. We characterized these memories in larval Drosophila melanogaster using a combination of Pavlovian conditioning, optogenetic activation of the dopaminergic central-brain DAN-i1864 neuron, and high-resolution video-tracking. This reveals their dependency on the number of training trials and the duration of DAN-i1864 activation, their temporal stability, and the parameters of locomotion that are modulated during memory expression. Together with previous results on 'punishment' versus 'relief' learning by DAN-f1 neuron activation, this reveals a 2×2 matrix of timing-dependent memory valence for the occurrence/termination of reward/punishment. These findings should aid the understanding and modelling of how brains decipher the predictive, causal structure of events around a target reinforcing occurrence.

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila.

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Aversive teaching signals from individual dopamine neurons in larval Drosophila show qualitative differences in their temporal "fingerprint".

Dopamine serves many functions, and dopamine neurons are correspondingly diverse. We use a combination of optogenetics, behavioral experiments, and high-resolution video-tracking 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.

Rewarding compounds identified from the medicinal plant Rhodiola rosea.

Preparations of Rhodiola rosea root are widely used in traditional medicine. They can increase life span in worms and flies, and have various effects related to nervous system function in different animal species and humans. However, which of the compounds in R. rosea is mediating any one of these effects has remained unknown in most cases. Here, an analysis of the volatile and non-volatile low-molecular-weight constituents of R. rosea root samples was accompanied by an investigation of their behavioral impact on Drosophila melanogaster larvae. Rhodiola rosea root samples have an attractive smell and taste to the larvae, and exert a rewarding effect. This rewarding effect was also observed for R. rosea root extracts, and did not require activity of dopamine neurons that mediate known rewards such as sugar. Based on the chemical profiles of R. rosea root extracts and resultant fractions, a bioactivity-correlation analysis (AcorA) was performed to identify candidate rewarding compounds. This suggested positive correlations for - among related compounds - ferulic acid eicosyl ester (FAE-20) and ß-sitosterol glucoside. A validation using these as pure compounds confirmed that the correlations were causal. Their rewarding effects can be observed even at low micromolar concentrations and thus at remarkably lower doses than for any known taste reward in the larva. We discuss whether similar rewarding effects, should they be observed in humans, would indicate a habit-forming or addictive potential.

Identification of Dopaminergic Neurons That Can Both Establish Associative Memory and Acutely Terminate Its Behavioral Expression.

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.SIGNIFICANCE STATEMENT In the struggle for survival, animals have to explore their environment in search of food. Once food is found, however, it is adaptive to prioritize exploiting it over continuing a search that would now be as pointless as searching for the glasses you are wearing. This exploration-exploitation trade-off is important for animals and humans, as well as for technical search devices. We investigate which of the only 10,000 neurons of a fruit fly larva can tip the balance in this trade-off, and identify a single dopamine neuron called DAN-i1 that can do so. Given the similarities in dopamine neuron function across the animal kingdom, this may reflect a general principle of how search is terminated once it is successful.

A biographical sketch of Troy D. Zars (1967-2018).

Troy D. Zars (1967-2018) was an American biologist. He studied the relationships between genes, neuronal circuits and behavior in the fruit fly Drosophila melanogaster. Zars co-pioneered the use of transgene expression to locally restore gene function in memory-defective fly mutants, an approach that provided breakthrough insights into the localization of memory traces in the fly brain. With ensuing refinements of the methods of transgene expression and the broadening in the range of transgenes to be expressed, this shaped the field of modern behavioral neurogenetics.

Recurrent architecture for adaptive regulation of learning in the insect brain.

Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. We provide a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. We discover afferent sensory pathways and a large population of neurons that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). We combine this with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. We find that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. Our study provides the most detailed view to date of biological circuit motifs that support associative learning.

An amino-acid mixture can be both rewarding and punishing to larval Drosophilamelanogaster.

Amino acids are important nutrients for animals because they are necessary for protein synthesis in particular during growth, as well as for neurotransmission. However, little is known about how animals use past experience to guide their search for amino-acid-rich food. We reasoned that the larvae of Drosophila melanogaster are suitable for investigating this topic because they are the feeding and growth stages in the life cycle of these holometabolous insects. Specifically, we investigated whether experiencing an odour with a 20 amino-acid mixture as a semi-natural tastant during training establishes odour-tastant associative memories. Across a broad concentration range (0.01-20 mmol l-1), such an amino-acid mixture was found to have a rewarding effect, establishing appetitive memory for the odour. To our surprise, however, manipulation of the test conditions revealed that relatively high concentrations of the amino-acid mixture (3.3 mmol l-1 and higher) in addition establish aversive memory for the odour. We then characterized both of these oppositely valenced memories in terms of their dependency on the number of training trials, their temporal stability, their modulation through starvation and the specific changes in locomotion underlying them. Collectively, and in the light of what is known about the neuronal organization of odour-food memory in larval D. melanogaster, our data suggest that these memories are established in parallel. We discuss the similarity of our results to what has been reported for sodium chloride, and the possible neurogenetic bases for concentration-dependent changes in valence when these tastants are used as reinforcers.

Reversal learning in Drosophila larvae.

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.

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

An optogenetic analogue of second-order reinforcement in Drosophila.

In insects, odours are coded by the combinatorial activation of ascending pathways, including their third-order representation in mushroom body Kenyon cells. Kenyon cells also receive intersecting input from ascending and mostly dopaminergic reinforcement pathways. Indeed, in Drosophila, presenting an odour together with activation of the dopaminergic mushroom body input neuron PPL1-01 leads to a weakening of the synapse between Kenyon cells and the approach-promoting mushroom body output neuron MBON-11. As a result of such weakened approach tendencies, flies avoid the shock-predicting odour in a subsequent choice test. Thus, increased activity in PPL1-01 stands for punishment, whereas reduced activity in MBON-11 stands for predicted punishment. Given that punishment-predictors can themselves serve as punishments of second order, we tested whether presenting an odour together with the optogenetic silencing of MBON-11 would lead to learned odour avoidance, and found this to be the case. In turn, the optogenetic activation of MBON-11 together with odour presentation led to learned odour approach. Thus, manipulating activity in MBON-11 can be an analogue of predicted, second-order reinforcement.

One-trial learning in larval Drosophila.

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 one-trial 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.

Rhodiola rosea root extract has antipsychotic-like effects in rodent models of sensorimotor gating.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant arctic root (Rhodiola rosea, L.) is growing in northern regions of Europe, Asia and North America. Extracts of R. rosea are used in traditional medicine for various conditions related to nervous system function. According to scientific studies from the last decades, the plant might have potential for use in the treatment of memory impairments, stress and depression, but reports concerning other neuropsychiatric disorders are scarce. AIM OF THE STUDY: In this context, our study aimed to examine potential antipsychotic-like effects of R. rosea root extract. MATERIALS AND METHODS: We tested the effects of R. rosea root extract on prepulse inhibition in rats and mice. Prepulse inhibition is an established operational measure of sensorimotor gating, which is impaired in schizophrenia and other psychotic disorders. RESULTS: R. rosea root extract increased prepulse inhibition in rats and mice. Interestingly, the R. rosea extract had stronger effects in those individual animals that had low baseline levels of prepulse inhibition. Therefore, we performed further experiments in which we pharmacologically induced a prepulse inhibition deficit by two different psychostimulants, either the dopamine D2 receptor agonist apomorphine or the NMDA receptor antagonist dizocilpine (MK-801). Pre-treatment with the R. rosea extract significantly restored both, apomorphine- and dizocilpine-induced prepulse inhibition deficits. CONCLUSIONS: The present study demonstrates that R. rosea extract robustly reverses prepulse inhibition deficits in rodents. This suggests antipsychotic-like effects of R. rosea extract. Future studies should focus on the pharmacological mechanisms underlying these effects.