Biology subjects

Ducrot, C.

Publications and source records attributed to Ducrot, C..

10 recordsLinked to original sources

bioRxiv2026

Hindbrain explants enable multimodal and longitudinal analysis of the developing olivo-cerebellar circuit at single-cell resolution

Experimental models that preserve native mammalian CNS circuitry while enabling longitudinal analysis of circuit assembly at single-cell resolution remain scarce, limiting mechanistic studies and therapeutic discovery. Here, we establish embryonic mouse hindbrain explants as a scalable in vitro model that maintains the long-range olivo-cerebellar circuit while providing direct experimental access to both pre- and postsynaptic neurons. The preparation supports repeated live imaging, targeted single-cell manipulation and labelling, electrophysiology, ultrastructural analysis, and single-cell RNA sequencing during circuit assembly. Hindbrain explants faithfully recapitulate key features of olivo-cerebellar organization and development, including cytoarchitecture, synaptic organization and maturation, neuronal differentiation, and spontaneous network activity while preserving developmental glial features. By combining developmental and physiological fidelity with longitudinal multimodal accessibility, this resource bridges the gap between reductionist cultures and technically demanding in vivo approaches, providing a versatile and ethical model for investigating the molecular and cellular mechanisms of cerebellar circuit assembly and disease.

neuroscience↗

bioRxiv2026

Neurospheres from primary rodent brain cells to probe the 3D organization and function of synapses

To improve our understanding of synapse assembly, there is a need for robust, easy-to-use, and physiologically relevant in-vitro models allowing the controllable formation of neuronal contacts in a reasonable time, whose structure and function can be investigated using advanced microscopy. To address this challenge, we engineered 3D cultures from rodent dissociated hippocampal cells, that spontaneously assemble in low attachment U-bottom wells into compact spheroids of reproducible dimensions (100-300 microns), determined by the number of seeded cells. These neurospheres contain a mix of neurons and glial cells and grow over time in culture, through the combination of cell proliferation and neurite extension. Neurospheres were immunostained in fluid phase, and/or sparsely electroporated for the multi-color visualization of synaptic proteins. Neurons extend an elaborate network of axons and dendrites, forming within 2 weeks numerous excitatory and inhibitory synapses identified at the structural level by confocal and electron microscopy, and at the functional level by electrophysiology. Periodic calcium oscillations throughout neurospheres further highlight network activity. Finally, we demonstrate the potential of neurospheres to study synaptogenesis by modulating and visualizing the adhesion protein neuroligin-1. Overall, neurospheres represent a standardized and cost-effective system to study synapse structure and function at high resolution in 3D, that should be quite appealing to the cellular neurobiology community.

neuroscience↗

bioRxiv2025

A tripartite protein complex promotes DNA transport during natural transformation in firmicutes.

Natural genetic transformation is a conserved mechanism of bacterial horizontal gene transfer, which is directed entirely by the recipient cell and facilitates the acquisition of new genetic traits such as antibiotic resistance. Transformation proceeds via the capture of exogenous DNA, its internalisation in single strand form (ssDNA) and its integration into the recipient chromosome by homologous recombination. While the proteins involved in these steps have mainly been identified, the specific mechanisms at play remain poorly characterised. This study takes advantage of recent advances in structural modelling to explore the uptake of ssDNA during transformation. Using the monoderm human pathogen Streptococcus pneumoniae, we model a tripartite protein complex composed of the transmembrane channel ComEC, and two cytoplasmic ssDNA-binding proteins ComFA and ComFC. Using targeted mutation and transformation assays, we propose that pneumococcal ComEC features a narrow channel for ssDNA passage, and we show this channel is conserved in the diderm Helicobacter pylori. We identify key residues involved in protein-protein and protein-ssDNA interactions in the pneumococcal tripartite complex model and we show them to be crucial for transformation efficiency. Structural modelling reveals that this tripartite protein complex and its interaction with ssDNA are conserved in firmicutes. Overall, this study validates a tripartite complex required for the internalisation of ssDNA during transformation in firmicutes, providing new insights into the molecular mechanisms involved in this horizontal gene transfer mechanism central to bacterial adaptation. It also demonstrates the power of recent structural modelling techniques such as AlphaFold3 as hypothesis generators and guides for designing experiments. Significance statementNatural genetic transformation is a key mechanism of horizontal gene transfer, conserved in bacteria. The investigation of transmembrane channel ComEC and its interaction partners was previously hindered by the difficulty of manipulating these proteins experimentally. Thanks to state-of-the-art structural modelling with AlphaFold3 and subsequent thorough experimental validation of this model using targeted mutation and transformation assays, we demonstrate the importance of the ComEC/ComFA/ComFC complex for single strand DNA (ssDNA) uptake during natural transformation in the human pathogen Streptococcus pneumoniae. Similar models in several other species suggest a widely conserved organization of this complex in firmicutes. In addition, we demonstrate that the ComEC transmembrane channel is also crucial for ssDNA uptake during natural transformation in Helicobacter pylori.

microbiology↗

bioRxiv2024

High-affinity detection of endogenously biotinylated neuroligin-1 at excitatory and inhibitory synapses using a tagged knock-in mouse strain

Neuroligins (NLGNs) are important cell adhesion molecules mediating trans-synaptic contacts between neurons. However, the high-yield biochemical isolation and visualization of endogenous NLGNs have been hampered by the lack of efficient antibodies to these proteins. Thus, to reveal their sub-cellular distribution, binding partners, and synaptic function, NLGNs have been extensively manipulated using knock-down, knock-out, or over-expression approaches, overall leading to controversial results. As an alternative to the manipulation of NLGN expression level, we describe here the generation of a new transgenic mouse strain in which native NLGN1 was N-terminally tagged with a small biotin acceptor peptide (bAP) that can be enzymatically biotinylated by the exogenous delivery of biotin ligase. After showing that knock-in mice exhibit normal behavior as well as similar synaptic number, ultrastructure, transmission properties, and protein expression levels when compared to wild type counterparts, we exploited the fact that biotinylated bAP-NLGN1 can be selectively isolated or visualized using high-affinity streptavidin conjugates. Using immunoblotting and immunofluorescence, we show that bAP-NLGN1 binds both PSD-95 and gephyrin and distributes equally well at excitatory and inhibitory synapses, challenging the historical view that NLGN1 is exclusively localized at excitatory synapses. Using super-resolution fluorescence microscopy and electron microscopy, we further highlight that bAP-NLGN1 forms in the synaptic cleft a subset of nanodomains each containing a few NLGN1 dimers, while the number of nanodomains per synapse positively scales with the post-synapse size. Overall, our study not only provides a novel, extensively characterized transgenic mouse model which will be made available to the scientific community, but also an unprecedented view of the nanoscale organization of endogenous NLGN1.

neuroscience↗

bioRxiv2023

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks

DNA double-strand breaks (DSB) must be repaired to ensure genome stability. Crucially, DSB ends must be kept together for timely repair. In Saccharomyces cerevisiae, two poorly understood pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB ends. Another requires the conversion of DSB ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB ends, revealing a new function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a new role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end separation and promotes DSB repair, revealing a novel mode of action and role for cohesin in safeguarding genome integrity.

molecular biology↗

bioRxiv2021

Neurexins Regulate GABA Co-release by Dopamine Neurons

Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which glutamate or GABA are released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. Here we tested the hypothesis that the trans-synaptic cell adhesion molecules neurexins (Nrxns) regulate DA neuron neurotransmission. Conditional deletion of all Nrxns in DA neurons (DAT::Nrxns KO) showed that loss of Nrxns does not impair the basic development and ultrastructural characteristics of DA neuron terminals. However, loss of Nrxns caused an impairment of DA transmission revealed as a reduced rate of DA reuptake following activity-dependent DA release, decreased DA transporter levels, increased vesicular monoamine transporter expression, and impaired amphetamine-induced locomotor activity. Strikingly, electrophysiological recording revealed an increase of GABA co-release from DA neuron axons in the striatum of the KO mice. These findings suggest that Nrxns act as key regulators of DA neuron connectivity and DA-mediated functions. HighlightsO_LIThe study provides the first direct evidence of the role of neurexins in dopaminergic neurons. C_LIO_LIThe synaptic adhesion molecules, neurexins, are not required for maintaining the structure of dopamine neuron terminals. C_LIO_LINeurexins regulate dopaminergic neurotransmission through regulation of dopamine reuptake, impacting amphetamine-induced locomotion. C_LIO_LIDeletion of Nrxns in DA neurons causes a region-specific increase of GABA release by DA neurons. C_LI

neuroscience↗

bioRxiv2021

The calcium sensor synaptotagmin-1 is critical for phasic axonal dopamine release in the striatum and mesencephalon, but is dispensable for basic motor behaviors in mice

In Parkinsons disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

neuroscience↗

bioRxiv2021

Neonatal 6-OHDA lesion of the SNc induces striatal compensatory sprouting from surviving SNc dopaminergic neurons without VTA contribution

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) are uniquely vulnerable to neurodegeneration in Parkinsons disease (PD). We hypothesize that their large axonal arbor is a key factor underlying their vulnerability, due to increased bioenergetic, proteostatic and oxidative stress. In keeping with this model, other DAergic populations with smaller axonal arbors are mostly spared during the course of PD and are more resistant to experimental lesions in animal models. Aiming to improve mouse PD models, we examined if neonatal partial SNc lesions could lead to adult mice with fewer SNc DA neurons that are endowed with larger axonal arbors because of compensatory mechanisms. We injected 6-hydroxydopamine (6-OHDA) unilaterally in the SNc at an early postnatal stage at a dose selected to induce loss of approximately 50% of SNc DA neurons. We find that at 10- and 90-days after the lesion, the axons of SNc DA neurons show massive compensatory sprouting, as revealed by the proportionally smaller decrease in tyrosine hydroxylase (TH) in the striatum compared to the loss of SNc DA neuron cell bodies. The extent and origin of this axonal sprouting was further investigated by AAV-mediated expression of eYFP in SNc or ventral tegmental area (VTA) DA neurons of adult mice. Our results reveal that SNc DA neurons have the capacity to substantially increase their axonal arbor size and suggest that mice designed to have reduced numbers of SNc DA neurons could potentially be used to develop better mouse models of PD, with elevated neuronal vulnerability. Graphical abstract textO_LIWe describe a technique to induce the loss of approximately 50% of SNc DA neurons in neonate mice using unilateral intranigral 6-OHDA (left panel). C_LIO_LICompensatory axonal sprouting was observed in the striatum as early as 10 days following the lesion (at P15), with effects lasting until adulthood (P90). C_LIO_LIConditional AAV-mediated expression of eYFP (green) reveals SNc DA neurons, projecting to the dorsal striatum (middle panel), and not VTA DA neurons, projecting to the ventral striatum (right panel), as the main source of compensatory axonal sprouting. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/437271v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@5febf7org.highwire.dtl.DTLVardef@17cc625org.highwire.dtl.DTLVardef@b4d509org.highwire.dtl.DTLVardef@e496a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6- hydroxydopamine model of Parkinsons disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

neuroscience↗

bioRxiv2020

Dopaminergic neurons establish a distinctive axonal arbor with a majority of non-synaptic terminals

Chemical neurotransmission in the brain typically occurs through synapses, which are structurally and functionally defined as sites of close apposition between an axon terminal and a postsynaptic domain. Ultrastructural examinations of axon terminals established by monoamine neurons in the brain often failed to identify a similar tight pre- and postsynaptic coupling, giving rise to the concept of "diffuse" or "volume" transmission. Whether this results from intrinsic properties of such modulatory neurons remains undefined. Using an efficient co-culture model, we find that dopaminergic neurons establish an axonal arbor that is distinctive compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. Furthermore, while most dopaminergic varicosities express key proteins involved in exocytosis such as synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in a subset of dopaminergic terminals that are in proximity to a target cell. Irrespective of their structure, a majority of dopaminergic terminals were found to be active. Finally, we found that the presynaptic protein Nrxn-1SS4- and the postsynaptic protein NL-1AB, two major components involved in excitatory synapse formation, play a critical role in the formation of synapses by dopamine neurons. Taken together, our findings support the idea that dopamine neurons in the brain are endowed with a distinctive developmental program that leads them to adopt a fundamentally different mode of connectivity, compared to glutamatergic and GABAergic neurons involved in fast point-to-point signaling. SIGNIFICANCE STATEMENTMidbrain dopamine (DA) neurons regulate circuits controlling movement, motivation, and learning. The axonal connectivity of DA neurons is intriguing due to its hyperdense nature, with a particularly large number of release sites, most of which not adopting a classical synaptic structure. In this study, we provide new evidence highlighting the unique ability of DA neurons to establish a large and heterogeneous axonal arbor with terminals that, in striking contrast with glutamate and GABA neurons, actively avoid contact with the target cells. The majority of synaptic and non-synaptic terminals express proteins for exocytosis and are active. Finally, our finding suggests that, NL-1A+B and Nrxn-1SS4-, play a critical role in the formation of synapses by DA neurons.

neuroscience↗