Abstract It is widely believed that starvation favours the processing of food-related cues, a notion here called the ’adaptive specificity hypothesis’. Indeed, in Drosophila melanogaster starvation is required for appetitive odour-sugar but not for aversive odour-shock memory. Results from Gruber et al. (2013) and Meschi et al. (2024), however, suggest that starvation improves aversive short-term memory, too, challenging this hypothesis. We survey how starvation affects Drosophila associative olfactory short-term memory across 26 learning tasks. These tasks differ in the reinforcers and the amount of training, the life stage of the animals, in the predictive structure and associative timing of the task, in whether memory is expressed as an increase or decrease in odour preference, and in whether the learned behaviour is motivated by obtaining reward or avoiding/ escaping punishment. In adult flies, an improvement was observed for appetitive odour-sugar memories, whereas all tasks yielding aversive memory were unaffected. Strikingly, ’appetitive’ tasks that are not sugar-related, namely odour-shock extinction learning and punishment-relief associations, were either unaffected or even impaired, supporting the ’adaptive specificity hypothesis’. In contrast, in 5-day-old larvae sugar-related appetitive associations were compromised, and the same was observed, to varying degrees, in larvae starved one day earlier and for aversive quinine associations, challenging the ’adaptive specificity hypothesis’. Furthermore, we observed starvation-induced changes in locomotion and preference for a subset of the cues used in our study. Our results defy a simplistic interpretation in terms of the ’adaptive specificity hypothesis’ and call for case-by-case analyses of how starvation affects learning and behaviour.

Adhesion G protein-coupled receptors (aGPCRs) are expressed in all organs and are involved in various mechanobiological processes. They are heavily alternatively spliced, forecasting an extraordinary molecular structural diversity. Here, we uncovered the existence of unconventional single-transmembrane (1TM)-containing ADGRL/Cirl proteins devoid of the conventional GPCR layout (i.e., the 7TM signaling unit) in Drosophila. These 1TM proteins are made as a result of intron retention and provide an N-terminal fragment that acts as an interactor to allow Gαo-dependent signaling through conventional 7TM-containing Cirl isoforms encoded by the same gene. This molecular mechanism determines sensory precision of neurons in response to mechanical stimulation in vivo. This action mode of aGPCR provides a promising entry point for experimental and therapeutic approaches to intervene in aGPCR signaling and implicates alternative splicing as a physiological strategy to express a given aGPCR together with its molecular interactor.

SUMMARYAdhesion G protein-coupled receptors (aGPCR) function as metabotropic mechanosensors in the nervous system and other organs. aGPCR are heavily spliced forecasting an extraordinary molecular structural diversity. Many predicted isoforms lack the transmembrane (7TM) signaling subunit, but to what extent these non-GPCR isoforms are produced and what physiological purpose they serve is unknown. Alternative splicing through intron retention ofADGRL/Latrophilin/CirlmRNA inDrosophilagenerates transcripts encoding unconventional proteins with an extracellular domain anchored by a single transmembrane helix (Cirl1TM). Here, we show thatCirl1TMtranscripts are translatedin vivoand that Cirl1TMbinds Cirl7TMN-terminal fragment-dependently. This interaction enables mechanosensory neurons to distinguish input intensities through Gαo-dependent signaling. Similarly, a direct interaction was found for mammalian GPR126/ADGRG6 isoforms. Together, our findings define intron retention and isoform-specific heteromerization as extraordinary molecular strategies to adjustCirl-dependent mechanosensation and demonstrate physiological relevance of versatile aGPCR isoform repertoire to tune cellular responsiveness.

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.

Excel file with source data, as well as precise sample sizes and results of statistical tests, of all figures and supplementary figures of this study. Each tab includes the data of one figure. Within each tab, the data are organised according to subfigures, with the data to the left and the sample sizes and statistical results to the right.

AbstractNeuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its numerically simple brain and neuromuscular system and its genetic accessibility, the larva of the fruit flyDrosophila melanogasteris an established model to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual animal, present studies of locomotion in larvalDrosophilamostly use group assays and measurements aggregated across individual animals. The alternative is to measure animals one at a time, an extravagance for larger-scale analyses. In principle or in practice, this in particular rules out grasping the inter- and intra-individual variability in locomotion and its genetic and neuronal determinants. Here we present the IMBA (Individual Maggot Behaviour Analyser) for tracking and analysing the behaviour of individual larvae within groups. Using a combination of computational modelling and statistical approaches, the IMBA reliably resolves individual identity across collisions. It does not require specific hardware and can therefore be used in non-expert labs. We take advantage of the IMBA first to systematically describe the inter- and intra-individual variability in free, unconstrained locomotion in wild-type animals. We then report the discovery of a novel, complex locomotion phenotype of a mutant lacking an adhesion-type GPCR. The IMBA further allows us to determine, at the level of individual animals, the modulation of locomotion across repeated activations of dopamine neurons. Strikingly, IMBA can also be used to analyse ‘silly walks’, that is patterns of locomotion it was not originally designed to investigate. This is shown for the transient backward locomotion induced by brief optogenetic activation of the brain-descending ‘mooncrawler’ neurons, and the variability in this behaviour. Thus, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and behavioural variability of individualDrosophilalarvae, with utility in multiple biomedical research contexts.

Finding food is a vital skill and a constant task for any animal, and associative learning of food-predicting cues gives an advantage in this daily struggle. The strength of the associations between cues and food depends on a number of parameters, such as the salience of the cue, the strength of the food reward and the number of joint cue-food experiences. We investigate what impact the strength of an associative odour-sugar memory has on the microbehaviour of Drosophila melanogaster larvae. We find that larvae form stronger memories with increasing concentrations of sugar or odour, and that these stronger memories manifest themselves in stronger modulations of two aspects of larval microbehaviour, the rate and the direction of lateral reorientation manoeuvres (so-called head casts). These two modulations of larval behaviour are found to be correlated to each other in every experiment performed, which is in line with a model that assumes that both modulations are controlled by a common motor output. Given that the Drosophila larva is a genetically tractable model organism that is well suited to the study of simple circuits at the single-cell level, these analyses can guide future research into the neuronal circuits underlying the translation of associative memories of different strength into behaviour, and may help to understand how these processes are organised in more complex systems.