Biology subjects

Landgraf, M.

Publications and source records attributed to Landgraf, M..

17 recordsLinked to original sources

bioRxiv2025

Neuronal excitability is permanently altered by activity manipulation during an embryonic critical period in Drosophila

Neuronal intrinsic excitability provides the baseline that homeostatic mechanisms act to preserve, yet the processes that establish a baseline remain poorly defined. Developmental critical periods (CPs) are thought to play a central role, but the link between early activity and long-term intrinsic properties is not well characterised. To address this, we used the genetic tractability of the Drosophila larval locomotor circuit to manipulate individual neurons during an embryonic CP. Following optogenetic excitation or inhibition, during the CP, we assessed intrinsic excitability of the same neurons in third-instar larvae (i.e. [~]5 days thereafter). We compared an excitatory premotor interneuron (A27h), an inhibitory premotor interneuron (A31k), and a motor neuron (aCC). Both interneurons exhibited anti-homeostatic responses: excitatory perturbation increased intrinsic excitability, while inhibitory perturbation decreased it, effects that persisted throughout larval development. In contrast, motor neurons showed no significant changes under the same conditions, revealing cell type-specific sensitivity to early activity. These findings build on the general principles of the functional relationships between CP activity and neuronal excitability and how intrinsic excitability is not passively set but actively shaped during these windows, with long-lasting, neuron-specific consequences. More broadly, our results highlight how developmental perturbations can alter the excitatory-inhibitory balance of mature neural circuits that may contribute to the aetiology of neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Maturation of GABAergic signalling times the opening of a critical period in Drosophila melanogaster

The occurrence of critical periods during the development of neural networks is widely documented. Activity manipulation when these periods are open can lead to permanent, and often debilitating, effects to the mature neural network. Detailed understanding of the specific contribution of critical periods to network development, however, remains elusive. This is partly because identified critical periods in mammals are present in complex sensory networks (e.g., visual and auditory) that make focused experimental manipulation challenging. It is significant, therefore, that critical periods have been identified in simpler model systems. A critical period occurs during the development of the embryonic locomotor network in the fruit fly, Drosophila melanogaster. Perturbation of neuronal activity during this period is sufficient to permanently destabilise the mature larval locomotor network: leaving it prone to induced seizures. Given a clear role of {gamma}-aminobutyric acid (GABA) in the timing of the mammalian critical period of ocular dominance, we sought to establish whether this neurotransmitter also regulates the opening of the Drosophila locomotor critical period. Utilising GABA agonists, antagonists, and genetics, we manipulated the embryonic GABAergic system and, at the end of larval life, measured an induced seizure phenotype in mature third-instar larvae. We found that potentiating GABAergic signalling, via embryonic exposure to diazepam or overexpression of the GABAA receptor rdl, induced precocious opening of the critical period. By contrast, exposure to the GABA antagonist gabazine, or knockdown of the GABA-synthetic enzyme Gad1, delayed opening. Thus, we show that critical period timing within the Drosophila CNS is dictated by GABAergic signalling, indicating a phylogenetically conserved role.

neuroscience↗

bioRxiv2025

Different mechanisms link gain and loss of kinesin functions to axonal degeneration

Axons are the slender, often meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organisms lifetime, meaning up to a century in humans. Long-term maintenance and sustained functionality of axons requires motor protein-driven transport distributing life-sustaining materials and organelles to places of need. It seems therefore plausible that loss of motor function would cause axon degeneration; however, also gain-of-function conditions were linked to disorders including motor neuron disease or spastic paraplegia. To understand this phenomenon, we studied [~]40 genetic manipulations of motor proteins, cargo linkers and regulators of reactive oxygen species in one standardised Drosophila primary neuron system. Using axonal microtubule bundle organisation as a relevant readout reflecting the state of axon integrity, we found that losses of Dynein heavy chain, KIF1A/Unc-104 and KIF5/Kinesin heavy chain (Khc) all cause bundle disintegration in the form of chaotically curled microtubules. Detailed functional studies of Khc and its adaptor proteins revealed that losses of mitochondrial or lysosomal transport cause ROS dyshomeostasis, which is a microtubule-curl-inducing condition in fly and mouse neurons alike. We find that hyper-activated Khc induces the same microtubule curling phenotype, not through ROS but likely more directly through enhanced mechanical forces. Studies with loss of Unc-104 or KIFBP and expression of an ALS-linked mutant form of the human Khc orthologue KIF5A suggest that loss or hyperactivation of different types of transport motors cause MT curling as a shared feature. We discuss a model which can explain our findings and their relevance for understanding motor-linked neurodegeneration.

neuroscience↗

bioRxiv2024

Conserved mitochondrial reactive oxygen species and HIF1-alpha signalling during an embryonic critical period directs subsequent nervous system development

As developing networks transition from spontaneous irregular to patterned activity, they undergo plastic tuning phases, termed "critical periods"; "critical" because disturbances during these phases can lead to lasting changes in network development and output. Critical periods are common to developing nervous systems, with analogous features shared from insects to mammals, yet the core signalling mechanisms that underly cellular critical period plasticity have remained elusive. To identify these, we exploited the Drosophila larval locomotor network as an advantageous model system. It has a defined critical period and offers unparalleled access to identified network elements, including the neuromuscular junction as a model synapse. We find that manipulations of a single motoneuron or muscle cell during the critical period lead to predictable, and permanent, cell-specific changes. This demonstrates that critical period adjustments occur at a single cell level. Mechanistically, we identified mitochondrial reactive oxygen species (ROS) as causative. Specifically, we show that ROS produced by complex I of the mitochondrial electron transport chain, generated by the reverse flow of electrons, are necessary and instructive for critical period-regulated plasticity. Downstream of ROS, we identified the Drosophila homologue of hypoxia inducible factor (HIF-1), as required for transducing the mitochondrial ROS signal to the nucleus. This signalling axis is also sufficient to cell autonomously specify changes in neuronal properties and animal behaviour but, again, only when activated during the embryonic critical period. Thus, we have identified specific mitochondrial ROS and HIF-1 as primary signals that mediate critical period plasticity.

developmental biology↗

bioRxiv2024

Heterogeneous responses to embryonic critical period perturbations among different components of the Drosophila larval locomotor circuit

As developing neural circuits become functional, they undergo a phase of heightened plasticity that facilitates network tuning in response to intrinsic and/or extrinsic stimuli. These developmental windows are termed critical periods (CPs), because perturbations during, but not outside, the CP can lead to lasting changes, such as the formation of sub-optimal or unstable networks. How separate, but connected elements, within a network might respond differently to a CP perturbation is not well understood. To study this, we used the locomotor network of the Drosophila larva as a model and heat stress as a CP stimulus that has ecological relevance. When embryos experienced heat stress the subsequent development of their locomotor network is changed, creating larvae with reduced crawling speed and decreased network stability. Developing body wall muscles and central neurons are sensitive to heat stress perturbations during distinct, consecutive phases of embryogenesis. Within the CNS, transient embryonic CP perturbation leads to increased synaptic drive from premotor interneurons to motoneurons, which in turn adopt reduced excitability. In contrast, the peripheral neuromuscular junction maintains normal synaptic transmission, despite significant structural changes. Thus, connected elements respond differentially to a CP perturbation, suggesting a sequence, or hierarchy, of network adjustment during the CP.

neuroscience↗

bioRxiv2023

Balance of Activity during a Critical Period Tunes a Developing Network

Developing neural circuits are influenced by activity and are especially sensitive to changes in activity during critical periods (CPs) of development. Changes occurring during a CP often become locked-in so that they affect the mature network. Indeed, several neurodevelopmental disorders have been linked to excessive activity during such periods. It is, therefore, important to identify those aspects of neural circuit development that are influenced by neural activity during a CP. In this study, we take advantage of the genetic tractability of Drosophila to show that activity perturbation during an embryonic CP permanently alters properties of the locomotor circuit. Specific changes we identify include increased synchronicity of motoneuron activity, and greater strengthening of excitatory over inhibitory synaptic drive to motoneurons. These changes are sufficient to reduce network robustness, evidenced by increased sensitivity to induced seizure. We also show that we can rescue these changes when increased activity is mitigated by inhibition provided by mechanosensory neurons. Similarly, we demonstrate a dose-dependent relationship between inhibition experienced during the CP, and the extent to which it is possible to rescue the hyperexcitable phenotype characteristic of the parabss mutation. This suggests that developing circuits must be exposed to a properly balanced sum of excitation and inhibition during the CP to achieve normal mature network function. Our results, therefore, provide novel insight into how activity during a CP shapes specific elements of a circuit, and how activity during this period is integrated to tune neural circuits to the environment in which they will likely function.

neuroscience↗

bioRxiv2022

Activity-regulated growth of motoneurons at the neuromuscular junction is mediated by NADPH oxidases

Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and Dual Oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.

neuroscience↗

bioRxiv2021

New cell biological explanations for kinesin-linked axon degeneration

Axons are the slender, up to meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organisms lifetime, meaning up to a century in humans. Axon maintenance requires life-sustaining motor protein-driven transport distributing materials and organelles from the distant cell body. It seems logic that impairing this transport causes systemic deprivation linking to axon degeneration. But the key steps underlying these pathological processes are little understood. To investigate mechanisms triggered by motor protein aberrations, we studied more than 40 loss- and gain-of-function conditions of motor proteins, cargo linkers or further genes involved in related processes of cellular physiology. We used one standardised Drosophila primary neuron system and focussed on the organisation of axonal microtubule bundles as an easy to assess readout reflecting axon integrity. We found that bundle disintegration into curled microtubules is caused by the losses of Dynein heavy chain and the Kif1 and Kif5 homologues Unc-104 and Kinesin heavy chain (Khc). Using point mutations of Khc and functional loss of its linker proteins, we studied which of Khcs sub-functions might link to microtubule curling. One cause was emergence of harmful reactive oxygen species through loss of Milton/Miro-mediated mitochondrial transport. In contrast, loss of the Kinesin light chain linker caused microtubule curling through an entirely different mechanism appearing to involve increased mechanical challenge to microtubule bundles through de-inhibition of Khc. The wider implications of our findings for the understanding of axon maintenance and pathology are discussed.

cell biology↗

bioRxiv2021

High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community

Endogenous starter cultures are used in the production of several cheeses around the world, such as Parmigiano-Reggiano, in Italy, Epoisses, in France, and Canastra, in Brazil. These microbial communities are responsible for many of the intrinsic characteristics of each of these cheeses. Bacteriophages are ubiquitous around the world, well known to be involved in the modulation of complex microbiological processes. However, little is known about phage-bacteria growth dynamics in cheese production systems, where phages are normally treated as problems, as the viral infections can negatively affect or even eliminate the starter culture during production. Furthermore, a recent metagenomic based meta-analysis has reported that cheeses contain a high abundance of phage-associated sequences. Here, we analyse the viral and bacterial metagenomes of Canastra cheese, a tradition artisanal cheese produced using an endogenous starter culture. We observe a very high phage diversity level, mostly composed of novel sequences. We detect several metagenomic assembled bacterial genomes at strain level resolution, and several putative phage-bacteria interactions, evidenced by the recovered viral and bacterial genomic signatures. We postulate that at least one bacterial strain detected could be endogenous to the Canastra region, in Brazil, and that its growth seems to be modulated by native phages present in this artisanal production system. This relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implication for all cheeses that employ similar production processes around the world.

microbiology↗

bioRxiv2020

NADPH oxidase generated reactive oxygen species and aquaporin conduits mediate activity-regulated dendritic plasticity.

Neurons utilize plasticity of dendritic arbors as part of a larger suite of adaptive plasticity mechanisms. This explicitly manifests with motoneurons in the Drosophila embryo and larva, where dendritic arbors are exclusively postsynaptic and are used as homeostatic devices, compensating for changes in synaptic input through adapting their growth and connectivity. We recently identified reactive oxygen species (ROS) as novel plasticity signals instrumental in this form of dendritic adjustment. ROS correlate with levels of neuronal activity and negatively regulate dendritic arbor size. Here, we investigated NADPH oxidases as potential sources of such activity-regulated ROS and implicate Dual Oxidase (but not Nox), which generates hydrogen peroxide extracellularly. We further show that the aquaporins Bib and Drip, but not Prip, are required for activity-regulated ROS-mediated adjustments of dendritic arbor size in motoneurons. These results suggest a model whereby neuronal activity leads to activation of the NADPH oxidase Dual Oxidase, which generates hydrogen peroxide at the extracellular face; aquaporins might then act as conduits that are necessary for these extracellular ROS to be channeled back into the cell where they negatively regulate dendritic arbor size.

neuroscience↗

bioRxiv2020

Electrophysiological validation of premotor interneurons monosynaptically connected to the aCC motoneuron in the Drosophila larval CNS.

Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC). Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use.

neuroscience↗

bioRxiv2020

Connectomics analysis reveals first, second, and third order thermosensory and hygrosensory neurons in the adult Drosophila brain

Animals exhibit innate and learned preferences for temperature and humidity - conditions critical for their survival and reproduction. Here, we leveraged a whole adult brain electron microscopy volume to study the circuitry associated with antennal thermosensory and hygrosensory neurons, which target specific ventroposterior (VP) glomeruli in the Drosophila melanogaster antennal lobe. We have identified two new VP glomeruli, in addition to the five known ones, and the projection neurons (VP PNs) that relay VP information to higher brain centres, including the mushroom body and lateral horn, seats of learned and innate olfactory behaviours, respectively. Focussing on the mushroom body lateral accessory calyx (lACA), a known thermosensory neuropil, we present a comprehensive connectome by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. We find that a few lACA-associated mushroom body intrinsic neurons (Kenyon cells) solely receive thermosensory inputs, while most receive additional olfactory and thermo- or hygrosensory PN inputs in the main calyx. Unexpectedly, we find several classes of lACA-associated neurons that form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a general hub for thermosensory circuitry. For example, we find DN1 pacemaker neurons that link the lACA to the accessory medulla, likely mediating temperature-based entrainment of the circadian clock. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron that receives input mainly from dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor neurons in the nerve cord. (249) HIGHLIGHTSO_LITwo novel thermo/hygrosensory glomeruli in the fly antennal lobe C_LIO_LIFirst complete set of thermosensory and hygrosensory projection neurons C_LIO_LIFirst connectome for a thermosensory centre, the lateral accessory calyx C_LIO_LINovel third order neurons, including a link to the circadian clock C_LI

neuroscience↗

bioRxiv2019

Motor circuit function is stabilized during postembryonic growth by anterograde trans-synaptic Jelly Belly - Anaplastic Lymphoma Kinase signaling

The brain adapts to a changing environment or growing body size by structural growth and synaptic plasticity. Mechanisms studied to date promote synaptic growth between partner neurons, while negative counterparts that inhibit such interactions have so far remained elusive. Here, we investigate the role of Jeb-Alk signaling in coordinating motor circuit growth during larval stages of Drosophila. We quantify neuronal growth dynamics by intra-vital imaging, and synaptogenesis at nanometer resolution using endogenously labeled synaptic proteins, conditionally tagged with a fluorophore, and link changes in circuit anatomy with altered synaptic physiology and behavior. We find that loss of Jeb-Alk signaling leads to increased strengthening of synaptic excitation by developmental addition of additional postsynaptic but not pre-synaptic specializations. These changes ultimately lead to an epilepsy-like seizure behavior. We thus demonstrate that trans-synaptic anterograde Jeb-Alk signaling acts to stabilize developmental plasticity and circuit function, and that it does so specifically during postembryonic growth.

neuroscience↗

bioRxiv2019

Activity manipulation of an excitatory interneuron, during an embryonic critical period, alters network tuning of the Drosophila larval locomotor circuit

As nervous systems develop, activity perturbations during critical periods can lead to permanently altered network function. However, how activity perturbation influences individual synapses, the network response and the underlying signalling mechanisms are not well understood. Here, we exploit a recently identified critical period in the development of the Drosophila larval locomotor circuit to show that activity perturbation differentially affects individual and identified synaptic pairings. Remarkably, we further show that activity-manipulation of a selective excitatory interneuron is sufficient to fully recapitulate the effects induced by network-wide activity disturbance; indicative that some neurons make a greater contribution to network tuning. We identify nitric oxide (NO)-signalling as a potential mediator of activity-dependent network tuning during the critical period. Significantly, the effect of NO-signalling to network tuning is dictated by the prior activity state of the network. Thus, this study provides mechanistic insight that is currently lacking into how activity during a critical period tunes a developing network.

neuroscience↗

bioRxiv2019

Unravelling the virome in birch: RNA-Seq reveals a complex of known and novel viruses

High-throughput sequencing (HTS), combined with bioinformatics for de novo discovery and assembly of plant virus or viroid genome reads, has promoted the discovery of abundant novel DNA and RNA viruses and viroids. However, the elucidation of a viral population in a single plant is rarely reported. In five birch trees of German and Finnish origin exhibiting symptoms of birch leaf-roll disease (BRLD), we identified in total five viruses, among which three are novel. The number of identified virus variants in each transcriptome ranged from one to five. The novel species are genetically - fully or partially - characterized, they belong to the genera Carlavirus, Idaeovirus and Capillovirus and they are tentatively named birch carlavirus, birch idaeovirus, and birch capillovirus, respectively. The only virus systematically detected by HTS in symptomatic trees affected by the BRLD was the recently discovered birch leafroll-associated virus. The role of the new carlavirus in BLRD etiology seems at best weak, as it was detected only in one of three symptomatic trees. Continuing studies have to clarify the impact of the carlavirus to the BLRD. The role of the Capillovirus and the Idaeovirus within the BLRD complex and whether they influence plant vitality need to be investigated. Our study reveals the viral population in single birch trees and provides a comprehensive overview for the diversities of the viral communities they harbor.

molecular biology↗

bioRxiv2019

Synaptic specificity is collectively determined by partner identity, location and activity

Our nervous system is organized into circuits with specifically matched and tuned cell-to-cell connections that are essential for proper function. The mechanisms by which presynaptic axon terminals and postsynaptic dendrites recognize each other and establish the correct number of connections are still incompletely understood. Sperrys chemoaffinity hypothesis proposes that pre- and postsynaptic partners express specific combinations of molecules that enable them to recognize each other. Alternatively, Peters rule proposes that presynaptic axons and postsynaptic dendrites use non-partner-derived global positional cues to independently reach their target area, and once there they randomly connect with any available neuron. These connections can then be further refined by additional mechanisms based on synaptic activity. We used the tractable genetic model system, the Drosophila embryo and larva, to test these hypotheses and elucidate the roles of 1) global positional cues, 2) partner-derived cues and 3) synaptic activity in the establishment of selective connections in the developing nerve cord. We altered the position or activity of presynaptic partners and analyzed the effect of these manipulations on the number of synapses with specific postsynaptic partners, strength of functional connections, and behavior controlled by these neurons. For this purpose, we combined developmental live imaging, electron microscopy reconstruction of circuits, functional imaging of neuronal activity, and behavioral experiments in wildtype and experimental animals. We found that postsynaptic dendrites are able to find, recognize, and connect to their presynaptic partners even when these have been shifted to ectopic locations through the overexpression of receptors for midline guidance cues. This suggests that neurons use partner-derived cues that allow them to identify and connect to each other. However, while partner-derived cues are sufficient for recognition between specific partners and establishment of connections;; without orderly positioning of axon terminals by positional cues and without synaptic activity during embryonic development, the numbers of functional connections are altered with significant consequences for behavior. Thus, multiple mechanisms including global positional cues, partner-derived cues, and synaptic activity contribute to proper circuit assembly in the developing Drosophila nerve cord.

neuroscience↗

bioRxiv2016

Reactive Oxygen Species Regulate Activity-Dependent Neuronal Structural Plasticity

Neurons are inherently plastic, adjusting their structure, connectivity and excitability in response to changes in activity. How neurons sense changes in their activity level and then transduce these to structural changes remains to be fully elucidated. Working with the Drosophila larval locomotor network, we show that neurons use reactive oxygen species (ROS), metabolic byproducts, to monitor their activity. ROS signals are both necessary and sufficient for activity-dependent structural adjustments of both pre- and postsynaptic terminals and for network output, as measured by larval crawling behavior. We find the highly conserved Parkinsons disease-linked protein DJ-1{beta} acts as a redox sensor in neurons where it regulates pre- and postsynaptic structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. Neuronal ROS thus play an important physiological role as second messengers required for neuronal and network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.

neuroscience↗