Biology subjects

Kobler, O.

Publications and source records attributed to Kobler, O..

8 recordsLinked to original sources

bioRxiv2025

Individual dopaminergic neurons induce unique, yet overlapping combinations of behavioural modulations including safety learning, memory retrieval and acute locomotion

Two evolutionary highly conserved functions of dopamine are to carry "teaching" signals during associative learning and to control movement. In mammals and humans, these functions are generally thought to be produced by different populations of neurons. Here, we investigated in the larva of Drosophila melanogaster whether both these functions can be induced by the same individual dopaminergic neurons in the central brain. Focusing on the dopaminergic neurons of the DL1-cluster, we asked whether the optogenetic activation of individual neurons established associative punishment and/or safety memories, controlled the retrieval of the established memories, and acutely modulated locomotion. We found that each neuron had a unique, yet overlapping set of behavioural effects. Several individual neurons both established a memory and modulated acute locomotion by increasing the animals bending and decreasing its velocity. Our results demonstrate that individual dopaminergic neurons can fulfil a surprisingly broad range of functions in different behavioural contexts. Given the highly conserved roles of the dopaminergic system across the animal kingdom, this study raises the question whether a similarly diverse functionality can be found also in other animals, including humans.

neuroscience↗

bioRxiv2024

Specific presynaptic functions require distinct Drosophila Cav2 splice isoforms

The multiplicity of neural circuits that accommodate the sheer infinite number of computations conducted by brains requires diverse synapse and neuron types. At many vertebrate synapses release probability and other aspects of presynaptic function are tuned by different combinations of Cav2.1, Cav2.2, and Cav2.3 channels. By contrast, most invertebrate genomes contain only one Cav2 gene. The one Drosophila Cav2 homolog, cacophony (cac), localizes to presynaptic active zones (AZs) to induce synaptic vesicle release. We hypothesize that Drosophila cac functional diversity is enhanced by two specific exon pairs that are mutually exclusively spliced and not conserved in vertebrates, one in the voltage sensor and one in the intracellular loop containing the binding site(s) for Ca{beta} and G-protein {beta}{gamma} subunits. We test our hypothesis by combining opto- and electrophysiological with neuroanatomical approaches at a fast glutamatergic model synapse, the Drosophila larval neuromuscular junction. We find that alternative splicing in the voltage sensor affects channel activation voltage and is imperative for normal synapse function. Only the isoform with the higher activation voltage localizes to the presynaptic AZ and mediates evoked release. Removal of these cac splice isoforms renders fast glutamatergic synapses non-functional. By contrast, alternative splicing at the other alternative exon that encodes the intracellular loop between the first and the second homologous repeats does not affect cac presynaptic AZ localization, but it tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other exon reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number upon selective exon excision increases paired pulse ratios and the variability of synaptic depression during low frequency stimulation trains (1 and 10 Hz), and thus affects short term plasticity. Effects on short term plasticity can be rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.

neuroscience↗

bioRxiv2023

Actin Y53 Phosphorylation Regulates Somatic F-actin Radial Translocation to Promote Neuronal Polarization

How neurons accomplish the growth of an axon, and several dendrites sequentially is a cellular process not well understood. Here, we show that preferential somatic F-actin delivery to neurites inhibits neurite growth during axon formation. Thus, the neurite receiving less somatic F-actin is growing as an axon. During dendrites outgrowth, radial somatic F-actin translocation into all neurites is drastically diminished suggesting that cellular growth is restricted by the somatic F-actin flow into the growing neurites. Mechanistically, we report that radial somatic F-actin translocation is mediated via the Myosin II motor. Pharmacological inhibition of these molecular motors promotes neurite elongation and precludes F-actin translocation into neurites. Moreover, we show that actin phosphorylation at Y53, which promotes F-actin instability, is enriched selectively in some neurite shafts to exclude/minimize translocation of somatic F-actin only into those neurites. Therefore, enrichment of actin pY53 in the neurite shaft correlates with the longer neurite during axon formation. Accordingly, the Kinesin-1 motor domain, which accumulates transiently in neurites of stage 2 neurons and only in the emerging axon of stage 3 neurons, localizes preferentially in the neurite with the increased actin pY53 signal in the neurite shaft. Finally, we demonstrate that microtubule acetylation promotes actin phosphorylation, and these cytoskeleton modifications coexist in the longest neurite that grows as an axon. Collectively, our data support a model in which somatic F-actin translocation into undifferentiated neurites impedes growth counteracted by actin pY53, consequently, supporting neuronal polarization during axon formation.

cell biology↗

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↗

bioRxiv2021

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 multifluorescence 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-resolution 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.

neuroscience↗

bioRxiv2020

Centrosome-dependent microtubule organization sets the conditions for axon formation

Microtubule remodeling is critical during axon development when the more stable microtubules populate the axon. It is not completely understood, however, how this local cytoskeleton remodeling is coordinated. The centrosome, the main microtubule-organizing center (MTOC), has been suggested to be crucial for axon specification 1-5. Conversely, it was proposed that axon elongation is independent of centrosomal functions 6. Here we report that microtubule dynamics in early neurons follow a radial organization which establishes the conditions for the axon formation. Using high-resolution microscopy of early developing neurons, we demonstrate that few somatic acetylated microtubules are restricted near the centrosome. At later stages, however, acetylated microtubules spread out in the soma and concentrate in the growing axon. Furthermore, live-imaging of the microtubule plus-end binding protein EB3 in early differentiating neurons shows that growing microtubules have increased length and growth speed near the MTOC, suggesting local differences that might favor axon selection. Importantly, due to the lack of somatic stable/acetylated microtubules in early developing neurons, disruption of the F-actin cytoskeleton does not induce multiple axons, as it does at later stages of differentiation. Finally, we demonstrate that overexpression of the centrosomal protein 120 (Cep120), known for promoting microtubule acetylation and stabilization, induces multiple axons, while its downregulation decreases the content of proteins regulating microtubule dynamics and stability, hence hampering axon formation. Collectively, our data show that early centrosome-dependent microtubule organization contributes to axon formation.

cell biology↗

bioRxiv2019

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

Organelle positioning within neurites is required for proper neuronal function. In dendrites with their complex cytoskeletal organization, transport of organelles is guided by local specializations of the microtubule and actin cytoskeleton, and by coordinated activity of different motor proteins. Here, we focus on the actin cytoskeleton in the dendritic shaft and describe dense structures consisting of longitudinal and branched actin filaments. These actin patches are devoid of microtubules and are frequently located at the base of spines, or form an actin mesh around excitatory shaft synapses. Using lysosomes as an example, we demonstrate that the presence of actin patches has a strong impact on dendritic organelle transport, as lysosomes frequently stall at these locations. We provide mechanistic insights on this pausing behavior, demonstrating that actin patches form a physical barrier for kinesin-driven cargo. In addition, we identify myosin Va as an active tether which mediates long-term stalling. This correlation between the presence of actin meshes and halting of organelles could be a generalized principle by which synapses control organelle trafficking.

neuroscience↗

bioRxiv2018

Radial F-actin Organization During Early Neuronal Development

The centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center (Farina et al., 2016), raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here we report, using super-resolution microscopy and live-cell imaging, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoconversion/photoactivation experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin towards the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers; hence sustaining initial neuronal development.

neuroscience↗