Birgit Michels

Visual learning in individually assayed Drosophila larvae

SUMMARY An understanding of associative learning is facilitated if it can be analyzed in a simple animal like the fruit fly Drosophila. Here, we introduce the first visual associative learning paradigm for larval Drosophila; this is remarkable as larvae have an order of magnitude fewer neurons than adult flies. Larvae were subjected to either of two reciprocal training regimes: Light+/Dark- or Light-/Dark+. Subsequently, all larvae were individually tested for their preference between Light versus Dark. The difference between training regimes was therefore exclusively which visual situation was associated with which reinforcer; differences observed during the test thus reflected exclusively associative learning. For positive reinforcement (+) we used fructose (FRU), and for negative reinforcement (-) either quinine or sodium chloride (QUI, NaCl). Under these conditions, associative learning could be reproducibly observed in both wild-type strains tested. We then compared the effectiveness of training using differential conditioning, with both positive and negative reinforcement, to that using only positive or only negative reinforcement. We found that FRU only, but neither QUI nor NaCl, was in itself effective as a reinforcer. This is the first demonstration of appetitive learning in larval Drosophila. It is now possible to investigate the behavioral and neuronal organization of appetitive visual learning in this simple and genetically easy-to-manipulate experimental system.

The carrot, not the stick: appetitive rather than aversive gustatory stimuli support associative olfactory learning in individually assayed Drosophila larvae

The ability to learn is universal among animals; we investigate associative learning between odors and “tastants” in larval Drosophila melanogaster. As biologically important gustatory stimuli, like sugars, salts, or bitter substances have many behavioral functions, we investigate not only their reinforcing function, but also their response-modulating and response-releasing function. Concerning the response-releasing function, larvae are attracted by fructose and repelled by sodium chloride and quinine; also, fructose increases, but salt and quinine suppress feeding. However, none of these stimuli has a nonassociative, modulatory effect on olfactory choice behavior. Finally, only fructose but neither salt nor quinine has a reinforcing effect in associative olfactory learning. This implies that the response-releasing, response-modulating and reinforcing functions of these tastants are dissociated on the behavioral level. These results open the door to analyze how this dissociation is brought about on the cellular and molecular level; this should be facilitated by the cellular simplicity and genetic accessibility of the Drosophila larva.

Maggot learning and Synapsin function.

Summary Drosophila larvae are focused on feeding and have few neurons. Within these bounds, however, there still are behavioural degrees of freedom. This review is devoted to what these elements of flexibility are, and how they come about. Regarding odour–food associative learning, the emerging working hypothesis is that when a mushroom body neuron is activated as a part of an odour-specific set of mushroom body neurons, and coincidently receives a reinforcement signal carried by aminergic neurons, the AC-cAMP-PKA cascade is triggered. One substrate of this cascade is Synapsin, and therefore this review features a general and comparative discussion of Synapsin function. Phosphorylation of Synapsin ensures an alteration of synaptic strength between this mushroom body neuron and its target neuron(s). If the trained odour is encountered again, the pattern of mushroom body neurons coding this odour is activated, such that their modified output now allows conditioned behaviour. However, such an activated memory trace does not automatically cause conditioned behaviour. Rather, in a process that remains off-line from behaviour, the larvae compare the value of the testing situation (based on gustatory input) with the value of the odour-activated memory trace (based on mushroom body output). The circuit towards appetitive conditioned behaviour is closed only if the memory trace suggests that tracking down the learned odour will lead to a place better than the current one. It is this expectation of a positive outcome that is the immediate cause of appetitive conditioned behaviour. Such conditioned search for reward corresponds to a view of aversive conditioned behaviour as conditioned escape from punishment, which is enabled only if there is something to escape from – much in the same way as we only search for things that are not there, and run for the emergency exit only when there is an emergency. One may now ask whether beyond ‘value’ additional information about reinforcement is contained in the memory trace, such as information about the kind and intensity of the reinforcer used. The Drosophila larva may allow us to develop satisfyingly detailed accounts of such mnemonic richness – if it exists.

Genetic Distortion of the Balance between Punishment and Relief Learning in Drosophila

Abstract: An experience with electric shock can support two opposing kinds of behavioral effects: Stimuli that precede shock during training are subsequently avoided as predictors for punishment, whereas stimuli that follow shock during training are later on approached, as they predict relief. We show here, for the fruit fly Drosophila, that upon the loss of white-function, the balance between these two kinds of learning is distorted in favor of punishment learning: white1118 mutants show stronger punishment learning and weaker relief learning, as compared to wild type flies. Thus, white1118 mutants establish, overall, more “negative” memories for the shock experience. This only concerns the mnemonic effects of the shock; the immediate, reflexive responsiveness to shock remains unaltered. Also, learning about reward is apparently unaffected, both in adult and larval Drosophila. Prompted by the proposed function of the White protein as the transporter for biogenic amine precursors, we probed the brains of white1118 mutants for the amounts of biogenic amines (octopamine, tyramine, dopamine, and serotonin) by using high-perssure liquid chromatography coupled to mass spectrometry. Using this method, we found, however, no difference between white1118 and wild type files for any of the probed amines. In any event, analyses of how the white1118 mutation affects the balance between punishment and relief learning should provide a study case of how heritable distortions of such balance can come about. Finally, the effects of the white1118 mutation should be considered as a source of confound when using white as the “marker gene” in behavior-genetic analyses of any sort.

Behavioral and Synaptic Plasticity Are Impaired upon Lack of the Synaptic Protein SAP47

The synapse-associated protein of 47 kDa (SAP47) is a member of a phylogenetically conserved gene family of hitherto unknown function. In Drosophila, SAP47 is encoded by a single gene (Sap47) and is expressed throughout all synaptic regions of the wild-type larval brain; specifically, electron microscopy reveals anti-SAP47 immunogold labeling within 30 nm of presynaptic vesicles. To analyze SAP47 function, we used the viable and fertile deletion mutant Sap47156, which suffers from a 1.7 kb deletion in the regulatory region and the first exon. SAP47 cannot be detected by either immunoblotting or immunohistochemistry in Sap47156 mutants. These mutants exhibit normal sensory detection of odorants and tastants as well as normal motor performance and basic neurotransmission at the neuromuscular junction. However, short-term plasticity at this synapse is distorted. Interestingly, Sap47156 mutant larvae also show a 50% reduction in odorant–tastant associative learning ability; a similar associative impairment is observed in a second deletion allele (Sap47201) and upon reduction of SAP47 levels using RNA interference. In turn, transgenically restoring SAP47 in Sap47156 mutant larvae rescues the defect in associative function. This report thus is the first to suggest a function for SAP47. It specifically argues that SAP47 is required for proper behavioral and synaptic plasticity in flies—and prompts the question whether its homologs are required for proper behavioral and synaptic plasticity in other species as well.

Synapsin Determines Memory Strength after Punishment- and Relief-Learning

Adverse life events can induce two kinds of memory with opposite valence, dependent on timing: “negative” memories for stimuli preceding them and “positive” memories for stimuli experienced at the moment of “relief.” Such punishment memory and relief memory are found in insects, rats, and man. For example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock training (“forward conditioning” of the odor), whereas after shock-odor training (“backward conditioning” of the odor) they approach it. Do these timing-dependent associative processes share molecular determinants? We focus on the role of Synapsin, a conserved presynaptic phosphoprotein regulating the balance between the reserve pool and the readily releasable pool of synaptic vesicles. We find that a lack of Synapsin leaves task-relevant sensory and motor faculties unaffected. In contrast, both punishment memory and relief memory scores are reduced. These defects reflect a true lessening of associative memory strength, as distortions in nonassociative processing (e.g., susceptibility to handling, adaptation, habituation, sensitization), discrimination ability, and changes in the time course of coincidence detection can be ruled out as alternative explanations. Reductions in punishment- and relief-memory strength are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued both by acutely restoring Synapsin and by locally restoring it in the mushroom bodies of mutant flies. Thus, both punishment memory and relief memory require the Synapsin protein and in this sense share genetic and molecular determinants. We note that corresponding molecular commonalities between punishment memory and relief memory in humans would constrain pharmacological attempts to selectively interfere with excessive associative punishment memories, e.g., after traumatic experiences.

Chapter 5 – ‘Decision Making’ in Larval Drosophila

The brain is the organ of behavior organization. It structures the solution to the problem of what to do. This is complicated because usually we cannot be certain which behavior would be relatively the best. These processes, taking place between the moment when an uncertainty between behavioral options is recognized and the actual expression of behavior, we regard as ‘taking a decision.’ Such decision making needs to integrate (1) sensory input, (2) the current status reflecting evolutionary and individual history, (3) the available behavioral options, and (4) their expected outcomes. We focus on the decision to behaviorally express an associative memory trace—or not. After sketching the architecture of the chemobehavioral system in larval Drosophila, we present a working hypothesis of odor–taste associative memory trace formation and then discuss whether outcome expectations contribute to the organization of conditioned behavior. We argue that indeed conditioned olfactory behavior is organized according to its expected outcome, namely toward finding reward or escaping punishment, respectively. Conditioned olfactory behaviors are thus not responsive in nature but, rather, are actions expressed for the sake of the sought-for reward and the attempted relief. In addition to the organization of such outcome expectations, we discuss parametric features (‘axes’) of behavioral tasks that we believe bear upon the decision character of the underlying process and discuss whether these features can be found, or may reasonably be sought for, in larval Drosophila. It is argued that rather than trying to draw a line between behavioral processes that reflect decisions and those that are not, it is more useful to ask how strong the decision character of a given behavioral faculty is?

Synapsin is required to “boost” memory strength for highly salient events

Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the Drosophila genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function at the behavioral level. Task-relevant sensorimotor functions, however, remain intact in the absence of Synapsin. Using an odor-sugar reward associative learning paradigm in larval Drosophila, we show that memory scores in mutants lacking Synapsin (syn(97)) are lower than in wild-type animals only when more salient, higher concentrations of odor or of the sugar reward are used. Furthermore, we show that Synapsin is selectively required for larval short-term memory. Thus, without Synapsin Drosophila larvae can learn and remember, but Synapsin is required to form memories that match in strength to event salience-in particular to a high saliency of odors, of rewards, or the salient recency of an event. We further show that the residual memory scores upon a lack of Synapsin are not further decreased by an additional lack of the Sap47 protein. In combination with mass spectrometry data showing an up-regulated phosphorylation of Synapsin in the larval nervous system upon a lack of Sap47, this is suggestive of a functional interdependence of Synapsin and Sap47.

'Peer pressure' in larval Drosophila?

ABSTRACT Understanding social behaviour requires a study case that is simple enough to be tractable, yet complex enough to remain interesting. Do larval Drosophila meet these requirements? In a broad sense, this question can refer to effects of the mere presence of other larvae on the behaviour of a target individual. Here we focused in a more strict sense on ‘peer pressure’, that is on the question of whether the behaviour of a target individual larva is affected by what a surrounding group of larvae is doing. We found that innate olfactory preference of a target individual was neither affected (i) by the level of innate olfactory preference in the surrounding group nor (ii) by the expression of learned olfactory preference in the group. Likewise, learned olfactory preference of a target individual was neither affected (iii) by the level of innate olfactory preference of the surrounding group nor (iv) by the learned olfactory preference the group was expressing. We conclude that larval Drosophila thus do not take note of specifically what surrounding larvae are doing. This implies that in a strict sense, and to the extent tested, there is no social interaction between larvae. These results validate widely used en mass approaches to the behaviour of larval Drosophila.