Pavlovian Conditioning of Larval Drosophila: An Illustrated, Multilingual, Hands-On Manual for Odor-Taste Associative Learning in Maggots

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.

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“…A standard odor-FRU reward associative learning paradigm was used 95 – 97 (Fig. 3e ), adapted to facilitate the pre-screen.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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“…It is also more resource-friendly, requiring fewer Petri dishes, less agarose, and lower quantities of tastant substances, making it environmentally and financially preferable over the three-trial version. For teaching purposes in high school classroom settings or for undergraduate laboratory courses (Michels et al 2017), one-trial training with a training trial duration of 2.5 min and using single-odor absolute conditioning with fructose as the reward might therefore be the procedure of choice.…”

Section: Practical Implicationsmentioning

confidence: 99%

One-trial learning in larval Drosophila

et al. 2019

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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.

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“…We followed standard methods for a two-odour, reciprocal conditioning paradigm (Scherer et al, 2003;Neuser et al, 2005; for detailed protocols, see Gerber et al, 2010Gerber et al, , 2013Apostolopoulou et al, 2013;Michels et al, 2017). To take into account the small size of L1, we used smaller Petri dishes (55 mm diameter) than is standard for L3 (90 mm), filled with either only 1% agarose ('pure') or 1% agarose plus 2 mol l −1 fructose as a reward.…”

Section: Gustatory Preferencementioning

confidence: 99%

The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3Drosophilalarvae

Almeida-Carvalho

Berh

Braun

et al. 2017

Journal of Experimental Biology

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Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Olmpiad, we probed stage 1 larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages.

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Pavlovian Conditioning of Larval Drosophila: An Illustrated, Multilingual, Hands-On Manual for Odor-Taste Associative Learning in Maggots

Cited by 29 publications

References 58 publications

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

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

One-trial learning in larval Drosophila

The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3Drosophilalarvae

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