Biology subjects

Thum, A. S.

Publications and source records attributed to Thum, A. S..

14 recordsLinked to original sources

bioRxiv2026

Context makes the difference: Temporally Resolved Dopaminergic Teaching Signals Shape Associative Memory in Drosophila Larvae

Animals can adapt their behavioral responses to environmental cues by learning from experience. This ability relies on the formation and recall of memories that are shaped by beneficial or detrimental consequences and regulated by the dopaminergic system, which is highly conserved across insect species. In the Drosophila melanogaster larva, eight of total [~]120 dopaminergic neurons (DANs) innervate the mushroom body (MB), a key center for associative memory. This subset of DANs can be anatomically grouped into two clusters of four cells: the primary protocerebral anterior medial (pPAM) cluster, associated with reward signaling, and the dorsolateral 1 (DL1) cluster, associated with punishment. Such a functional dichotomy is observed in larval and adult Drosophila and reflects a fundamental organizational principle of reinforcement learning across invertebrate and even vertebrate species. Aversive reinforcement through high-salt exposure is encoded within the DL1 cluster in a combinatorial and heterogeneous manner, critically involving two neurons, DAN-f1 and DAN-g1. Using temporally precise optogenetic activation and inhibition during olfactory conditioning, we show that these neurons can modulate memory strength and valence. Their effects are most often consistent and predictable, enabling accurate computational modeling of DAN-driven teaching signals. By manipulating the intrinsic physiology of DAN-f1 and DAN-g1 and altering the valence of gustatory input, we are beginning to understand at the single-cell level how dopaminergic activity is systematically adjusted to control memory formation.

neuroscience↗

bioRxiv2026

The 3D micro- and nanostructure of the apical extracellular matrix in Drosophila

The apical extracellular matrix (aECM) serves as a dynamic scaffold, orchestrating tissue growth, cellular communication, and wound repair, and thus functions as a powerful biomaterial. In this study, we explored the intricate three-dimensional landscape of the Drosophila larval aECM using advanced 3D Volume electron microscopy. This technique opens a window into understanding and mimicking the diversity of aECM surfaces and architectures of the exoskeletal cuticles. We reconstructed the striking forms of denticles, hairs, and sensory organs, including double sensilla and Keils organs, at the epidermal surface. Our reconstructions brought the larval tracheal system and the sponge-like felt chamber (Filzkorper) of the spiracles into sharp relief, revealing a labyrinth of tiny cavities surrounding a central tube that extends from the posterior opening to the tracheal lumen. When examining the ultrastructure of transport-active anal pad cells, we discovered an inverted pattern of cuticular pore-canal structures, which supports their suggested role in osmoregulation across the aECM. Our findings further suggest that ordinary epidermal cells weave an intricate network of pore canals, enabling the orchestration of material distribution throughout the entire aECM. Moreover, we reconstructed tendon cells and chordotonal organs that form a branching network of numerous filamentous intracuticular fibers that weave through the aECM, linking the exoskeleton to muscles and sensory organs. By thoroughly mapping the intricate ultrastructural features of the aECM, this study opens new avenues for exploring how its protective, mechanical, and sensory roles intertwine, inspiring the next generation of tailor-made biomimetic materials.

developmental biology↗

bioRxiv2026

Octopamine receptors at a glance: from expression and anatomical maps to their role in development and behavior in the Drosophila melanogaster larva

Octopamine is involved in a variety of different physiological and behavioral mecha-nisms in Drosophila melanogaster. Throughout the life cycle of the fruit fly, from the larva to the adult, octopaminergic neurons in both the central and the peripheral nerv-ous system target a multitude of neurons and even non-neuronal tissues, making it challenging to analyze individual mechanisms of octopamine function. One approach to deconstructing this complex system is to examine the postsynaptic components of signal transmission. In Drosophila, octopamine interacts with six distinct G-protein-coupled receptors. For some of these receptors, expression maps and functional im-plications have been described. In contrast, other receptors have been neglected, partly due to the lack of suitable genetic tools. Here, for the first time, we compiled a complete set of mutant lines of all known octopamine receptors, all generated using the same genetic tool, the recently established Trojan Exon system. It integrates the Gal4/UAS binary expression strategy while simultaneously impairing receptor func-tion. This enabled us to generate a comprehensive anatomical map of receptor ex-pression in the larva and, at the same time, analyze the function of individual octopa-mine receptors during larval development, chemosensory perception and locomotion. All octopamine receptors (Oamb, Oct2R, Oct{beta}1R, Oct{beta}2R, Oct{beta}3R, and Oct-TyrR) showed extensive signal in the central nervous system. The same was found for the peripheral nervous system, with the exception of Oct{beta}2R, which showed pronounced expression in the somatic muscles. We also observed a previously undescribed role of Oct{beta}1R, Oct{beta}3R, and Oct-TyrR in larval hatching and in the survival of larvae and pupae. Molecular evaluation of the Trojan Exon octopamine lines supports our analy-sis. In addition, we combined the experimental results with gene expression data from the different development stages of Drosophila melanogaster and from different tis-sues and cell populations throughout the body. Overall, we compiled, analyzed and validated a complete set of octopamine lines which, together with gene expression analysis, provides a basis for further functional studies on the larval octopaminergic system.

neuroscience↗

bioRxiv2025

A switch in arousal circuit architecture shapes sleep across the lifespan

Sleep is a continuous behavior across the lifespan, yet its features and functions evolve markedly with development1-4. In Drosophila melanogaster, as in mammals, early life sleep differs from mature sleep5,6, but it is unknown whether disparate sleep regulatory mechanisms underlie these changes. Here, we identify distinct populations of octopaminergic (OA) neurons that promote arousal in larval and adult flies, thus revealing a developmental switch in sleep-wake circuit architecture. Of eight OA neurons present in the sub-esophageal zone (SEZ) of the nervous system at both life stages, dedicated, non-overlapping subsets drive arousal in larvae versus adults. Morphologic and connectomic analyses show that larval OA arousal neurons project primarily to the ventral nerve cord and lack substantial sensory input, suggesting a circuit logic optimized for internally driven arousal during early development. In contrast, adult OA arousal neurons target higher brain regions involved in cognition and receive rich multimodal sensory input, supporting wakefulness in response to environmental cues. These findings highlight a developmental transition in arousal circuitry that mirrors changing ecological demands, with juvenile systems organized to prioritize growth and feeding, insulated from sensory disturbance, and mature systems supporting sensory-guided behavior. Our results support a model of sleep regulation as a developmentally dynamic process, in which shared neuromodulators like OA operate through distinct cellular substrates tailored to life stage- specific behavioral priorities.

neuroscience↗

bioRxiv2025

Selective octopaminergic tuning of mushroom body circuits during memory formation

The catecholamines octopamine and tyramine undoubtedly have a major impact on the life of an insect. A wide range of physiological processes and behaviours are regulated by these neurotransmitters/hormones. Octopamine and tyramine act homologous to the adrenergic system of vertebrates, primarily adapting the organism to the given situation, by switching between the states of alertness and rest. Interestingly, higher brain functions like learning and memory are also regulated by octopamine and tyramine. About 30 years ago, initial work in Drosophila has demonstrated that dopaminergic neurons signal punishment, while octopaminergic neurons signal reward during olfactory associative learning and memory. In the meantime, however, it has become clear that distinct types of dopaminergic neurons convey both reward and punishment signals to the mushroom bodies, a central brain region responsible for the formation and storage of associative memories. Although some conflicting data remain, these findings challenge the previously established model of functional segregation and may limit the proposed role of octopamine neurons as teaching neurons during memory formation. We have therefore re-examined the role of octopamine in learning and memory in Drosophila larvae. Through a combination of Ca2+ imaging, anatomical studies and gain-of-function and loss-of-function behavioural approaches, we demonstrate that octopamine signalling plays a crucial role in larval learning by modulating dopaminergic neurons across distinct cell clusters to orchestrate memory processes.

neuroscience↗

bioRxiv2025

Morphology and ultrastructure of pharyngeal sense organs of Drosophila larvae

This study provides a comprehensive ultrastructural analysis of the pharyngeal sensory system in Drosophila melanogaster larvae, focusing on the four major pharyngeal sense organs: the ventral pharyngeal sensilla (VPS), dorsal pharyngeal sensilla (DPS), dorsal pharyngeal organ (DPO), and posterior pharyngeal sensilla (PPS). Our analysis revealed 15 sensilla across these organs, comprising four mechanosensory, nine chemosensory, and two dual-function sensilla. We identified 35 Type I neurons (six mechanosensory and 29 chemosensory) and six Type II neurons with putative chemosensory functions. Additional sensory structures, including papilla sensilla and chordotonal organs in the cephalopharyngeal region, were characterized. This detailed mapping and classification of pharyngeal sensory structures completes their structural characterization and provides a foundation for future anatomical and functional studies of sensory perception in insects. This work represents a significant step towards a complete analysis of the larval sensory system, providing new opportunities for investigating how an organism processes sensory information to navigate and interact with its environment.

neuroscience↗

bioRxiv2023

Drosophila Bchs overexpression recapitulates human WDFY3 neurodevelopmental phenotypes with implications for glial cell involvement in altered head circumference

The autophagy adaptor WDFY3 is linked to neurodevelopmental delay and altered brain size. Loss-of-function variants are associated with an increased brain size in both humans and mice. We thus, hypothesized that the microcephaly observed in some of the patients may be related to a gain-of-function of the WDFY3 gene product. While the role of WDFY3 loss-of-function has been studied extensively in neurons, little is known about the effects of WDFY3 overexpression in different neural cell types. We utilized a Drosophila melanogaster overexpression model to investigate the effect of the WDFY3 ortholog Bchs (blue cheese) on development, CNS size, and gene expression profiles. Glial and neuronal overexpression of Bchs impaired CNS development, locomotion and autophagy. Glial overexpression of Bchs also altered CNS size significantly. We identified 79 genes that were differentially expressed and overlapped in flies that overexpress Bchs in glial and neuronal cells, respectively. Additionally, upon neuronal Bchs overexpression differentially expressed genes clustered in gene ontology categories associated with autophagy and mitochondria. Our data indicate that WDFY3/Bchs overexpression in both neurons and glial cells results in impaired neural development, which corresponds to symptoms observed in WDFY3-related neurodevelopmental delay.

neuroscience↗

bioRxiv2023

Morphology and ultrastructure of external sense organsof Drosophila larvae

Sensory perception is the ability through which an organism is able to process sensory stimuli from the environment. This stimulus is transmitted from the peripheral sensory organs to the central nervous system, where it is interpreted. Drosophila melanogaster larvae possess peripheral sense organs on their head, thoracic, and abdominal segments. These are specialized to receive diverse environmental information, such as olfactory, gustatory, temperature or mechanosensory signals. In this work, we complete the description of the morphology of external larval sensilla and provide a comprehensive map of the ultrastructure of the different types of sensilla that comprise them. This was achieved by 3D electron microscopic analysis of partial and whole body volumes, which contain high-resolution and complete three-dimensional data of the anatomy of the sensilla and adjacent ganglia. Our analysis revealed three main types of sensilla on thoracic and abdominal segments: the papilla sensillum, the hair sensillum and the knob sensillum. They occur solitary or organized in compound sensilla such as the thoracic keilins organ or the terminal sensory cones. We present a spatial map defining these sensilla by their position on thoracic and abdominal segments. Further, we identify and name the sensilla at the larval head and the last fused abdominal segments. We show that mechanosensation dominates in the larval peripheral nervous system, as most sensilla have corresponding structural properties. The result of this work, the construction of a complete structural and neuronal map of the external larval sensilla, provides the basis for following molecular and functional studies to understand which sensory strategies the Drosophila larva employs to orient itself in its natural environment.

neuroscience↗

bioRxiv2023

Four Individually Identified Paired Dopamine Neurons Signal Taste Punishment in Larval Drosophila

Dopaminergic neurons (DANs) carry out multiple tasks in the brain, including the transmission of information related to rewards and punishments across various animal species. They are responsible for evaluating sensory input, storing resulting associations as memory, and continuously updating them based on their relevance and reliability. Accurate comprehension of the dopaminergic systems operation necessitates an understanding of the specific functions mediated by individual DANs. To this end, our research employs Drosophila larvae, which possess approximately 12,000 neurons in their brains, of which only around 1% (approximately 120) are DANs. The presynaptic projections to the mushroom body (MB) - a brain region pivotal for associative olfactory learning in insects - are limited to only eight larval dopaminergic neurons. These DANs are further subdivided into two clusters: the primary protocerebral anterior medial cluster (pPAM) comprises four cells, and the dorsolateral 1 cluster (DL1) comprises the remaining four cells. Our findings confirm previous research that demonstrates that the pPAM DANs innervating the MBs medial lobe encode for a gustatory sugar reward signal. Furthermore, we have identified four DANs in the DL1 cluster - DAN-c1, DAN-d1, DAN-f1, and DAN-g1 - each of which innervates distinct compartments of the MB peduncle, lateral appendix, and vertical lobe. Optogenetic activation of DAN-f1 and DAN-g1 alone suffices to substitute for punishment. Furthermore, optogenetic inhibition, calcium imaging results and electron microscopy-based reconstruction of all sensory input circuits to the four DL1 DANs demonstrate that each DAN encodes a different aspect of punishment, with DAN-g1 being of central importance for the salt dependent teaching signal. To summarize, our investigation has revealed the existence of a cellular division of labor among larval DANs concerning the transmission of dopaminergic reward (pPAM cluster) and punishment signals (DL1 cluster). Individual DANs in each cluster encode for distinct but partially overlapping aspects of the teaching signal. The striking resemblance in the organizing principle of larval DANs with that of its adult counterpart and the mammalian basal ganglion suggests that there may be a limited number of efficient neural circuit solutions available to address more complex cognitive challenges in nature.

animal behavior and cognition↗

bioRxiv2022

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 statementThe 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.

neuroscience↗

bioRxiv2022

Developmental emergence of sleep rhythms enables long-term memory capabilities in Drosophila

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day (1-4). The mechanism through which sleep rhythms emerge, and the consequent advantage conferred to a juvenile animal, are unknown. In 2nd instar Drosophila larvae (L2), like human infants, sleep is not under circadian control (5). Here, we identify the precise developmental timepoint when the circadian clock begins to regulate sleep in Drosophila, leading to the emergence of sleep rhythms at the early 3rd instar stage (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousalpromoting Dh44 neurons, bringing arousal under clock control to drive the emergence of circadian sleep. Finally, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of an aversive cue, and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

neuroscience↗

bioRxiv2021

Ethanol-guided behavior in Drosophila larvae

Chemosensory signals allow vertebrates and invertebrates not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. Ethanol is a substance found in the natural environment of Drosophila melanogaster. Accordingly, D. melanogaster has evolved specific sensory systems, physiological adaptations, and associated behaviors at its larval and adult stage to perceive and process ethanol. To systematically analyze how D. melanogaster larvae respond to naturally occurring ethanol, we examined ethanol-induced behavior in great detail by parametrically reevaluating existing approaches and comparing them with new experiments. Using behavioral assays, we confirm that larvae are attracted to different concentrations of ethanol in their environment. This behavior is controlled both by olfactory and contact cues. It is independent of previous exposure to ethanol in their food. Moreover, moderate, naturally occurring ethanol concentration of 4% results in increased larval fitness. On the contrary, higher concentrations of 10% and 20% ethanol, which rarely or never appear in nature, increase larval mortality. Finally, ethanol also serves as a positive teaching signal in learning and memory and updates valence associated with simultaneously processed odor information. Since information on how larvae perceive and process ethanol at the genetic and neuronal level is limited, the establishment of standardized assays described here is an important step towards their discovery.

animal behavior and cognition↗

bioRxiv2019

Multilevel feedback architecture for adaptive regulation of learning in the insect brain

Modulatory (e.g. dopaminergic) neurons provide \"teaching signals\" that drive associative learning across the animal kingdom, but the circuits that regulate their activity and compute teaching signals are still poorly understood. We provide the first synaptic-resolution connectome of the circuitry upstream of all modulatory neurons in a brain center for associative learning, the mushroom body (MB) of the Drosophila larva. We discovered afferent pathways from sensory neurons, as well as an unexpected large population of 61 feedback neuron pairs that provide one- and two-step feedback from MB output neurons. The majority of these feedback pathways link distinct memory systems (e.g. aversive and appetitive). We functionally confirmed some of the structural pathways and found that some modulatory neurons compare inhibitory input from their own compartment and excitatory input from compartments of opposite valence, enabling them to compute integrated common-currency predicted values across aversive and appetitive memory systems. This architecture suggests that the MB functions as an interconnected ensemble during learning and that distinct types of previously formed memories can regulate future learning about a stimulus. We developed a model of the circuit constrained by the connectome and by the functional data which revealed that the newly discovered architectural motifs, namely the multilevel feedback architecture and the extensive cross-compartment connections, increase the computational performance and flexibility on learning tasks. Together our study provides the most detailed view to date of a recurrent brain circuit that computes teaching signals and provides insights into the architectural motifs that support reinforcement learning in a biological system.

neuroscience↗

bioRxiv2017

The Complete Connectome Of A Learning And Memory Center In An Insect Brain

Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higherorder circuit supporting associative memory has not been previously available. We reconstructed one such circuit at synaptic resolution, the Drosophila larval mushroom body, and found that most Kenyon cells integrate random combinations of inputs but a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections between output neurons could enhance the selection of learned responses. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory center.

neuroscience↗