Biology subjects

Dieudonne, S.

Publications and source records attributed to Dieudonne, S..

7 recordsLinked to original sources

bioRxiv2026

Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

The advent of optogenetics and the rapid development of genetically encoded actuators and reporters for calcium, voltage, neurotransmitters, and other molecules have revolutionized neuroscience research by enabling precise, non-invasive optical interrogation of neural circuits. Successful implementation, however, critically depends on efficient reporter expression in defined neuronal populations. We present a Gateway(R)-based cloning platform providing a robust pipeline for rapid, efficient construction of recombinant adeno-associated virus (rAAV) vectors optimized for neuroscience applications. This platform addresses promoter selection, indicator engineering, and capsid-type optimization, and enables systematic optimization of vector components across a range of optical tools. The benefit of this modular approach is illustrated in the case of ULoVE, a two-photon microscopy method based on acousto-optic deflectors (AODs). This method provides serial light-targeting with kHz sampling rates and high signal-to-noise ratio in vivo, which imposes stringent requirements on indicator expression -- including cell-type specificity, sparse labelling, and precise control of expression levels-- efficiently met through the combinatorial flexibility of the Gateway pipeline. As specific examples, Gateway-constructed Cre-driver viruses combined with Cre-dependent reporters enabled cell-type-specific labelling for two complementary applications. In cerebellar Purkinje cells, an L7::Cre driver virus paired with a Gateway-constructed voltage indicator (JEDI2P-Kv) and ULoVE two-photon excitation at ~5 kHz enables resolution of sub-millisecond dendritic voltage dynamics in awake, behaving mice, including optical detection of dendritic spikelets previously accessible only via intracellular electrophysiology. The same driver virus paired with calcium indicators (GCaMP6f, jRGECO1a) resolves climbing fiber-evoked calcium kinetics under the same conditions. In acute cerebellar slices, a kit::Cre driver virus enabled cell-type-specific expression of the optogenetic actuator ChR2(H134R) in molecular layer interneurons, where ULoVE doughnut-pattern photostimulation achieved single-cell, sub-millisecond optogenetic activation with micron-scale spatial resolution. Beyond ULoVE applications, the same sparse, strong labelling also proved suitable for anatomical tracing of axonal projections in cleared cerebellar tissue, resolving individual DCN axon terminals at the granule cell layer --molecular layer boundary. Taken together, our dual-front approach --combining a modular AAV expression pipeline with AOD-based optical acquisition --provides an integrated platform for developing sophisticated optical tools, including but not limited to AOD-based microscopy, for neural circuit interrogation.

neuroscience↗

bioRxiv2024

A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

neuroscience↗

bioRxiv2024

Natural variation suggests new mechanisms for bract development in Arabidopsis, desynchronising bract suppression from the floral transition

Plant development may be viewed as a sequence of tightly orchestrated events in space and time. The coordination of developmental stages gives rise to various organs, such as leaves or flowers. For instance, in Arabidopsis thaliana, leaf/bract development ceases once the first flower emerges. Unravelling the mechanisms of this regulation is, therefore, key for understanding how developmental programs are coordinated during the floral transition. In this study, we take advantage of a previously overlooked natural variation that desynchronizes bract repression from the initiation of the first flowers. We found that prolonged bract formation after the floral transition depends on complex genetic interactions, involving at least four loci. Surprisingly, none of these loci contained the floral identity genes previously implicated in bract repression, suggesting that other genes are involved in the synchronisation of bract and flower programs. Furthermore, although ectopic bracts in inflorescences have been interpreted as a prolonged vegetative state, time-series transcriptomics and curve registration revealed a more complex scenario whereby many genes desynchronize in terms of expression between these developmental programs. As a consequence, transcriptional differences peak at the floral transition when bract development is prolonged, affecting a wide variety of biological processes not necessarily associated with vegetative state. A transient increase in transcriptome divergence has been proposed to account for morphological variation between species in animals and plants under the "inverse hourglass" model. Our results suggest that such a model could also explain the sensitivity of certain developmental transitions to phenotypic variation within species, as reported here for the floral transition.

plant biology↗

bioRxiv2023

Identification and organization of a postural anti-gravity module in the cerebellar vermis

The cerebellum is known to control the proper balance of isometric muscular contractions that maintain body posture. Current optogenetic manipulations of the cerebellar cortex output, however, have focused on ballistic body movements, examining movement initiation or perturbations. Here, by optogenetic stimulations of cerebellar Purkinje cells, the output of the cerebellar cortex, we evaluate body posture maintenance. By sequential analysis of body movement, we dissect the effect of optogenetic stimulation into a directly induced movement that is then followed by a compensatory reflex to regain body posture. We identify a module in the medial part of the anterior vermis which, through multiple muscle tone regulation, is involved in postural anti-gravity maintenance of the body. Moreover, we report an antero-posterior and medio-lateral functional segregation over the vermal lobules IV/V/VI. Taken together our results open new avenues for better understanding of the modular functional organization of the cerebellar cortex and its role in postural anti-gravity maintenance. HighlightsO_LIVermal Purkinje cell activation elicits a graded postural collapse in the standing mouse C_LIO_LIThe collapse triggers a secondary composite postural reflex C_LIO_LIAn identified cerebellar module is involved in postural anti-gravity tone maintenance C_LIO_LIThe anti-gravity function is somatotopically organized within this module C_LI

neuroscience↗

bioRxiv2022

Neurotransmitter content heterogeneity within an interneuron class shapes inhibitory transmission at a central synapse

Neurotransmitter content is deemed the most basic defining criterion for neuronal classes, contrasting with the intercellular heterogeneity of many other molecular and functional features. Here we show, in the adult mouse brain, that neurotransmitter content variegation within a neuronal class is a component of its functional heterogeneity. Most Golgi cells (GoCs), the well-defined class of cerebellar interneurons inhibiting granule cells (GrCs), contain cytosolic glycine, accumulated by the neuronal transporter GlyT2, and GABA in various proportions. To assess the functional consequence of this neurotransmitter variation, we paired GrCs recordings with optogenetic stimulations of single GoCs, which preserve the intracellular transmitter mixture. We show that the strength and decay kinetics of GrCs IPSCs, which are entirely mediated by GABAA receptors are negatively correlated to the presynaptic expression of GlyT2 by GoCs. We isolate a slow spillover component of GrCs inhibition that is also affected by the expression of GlyT2, leading to a 56 % decrease in relative charge. Acute manipulations of cytosolic GABA and glycine supply recapitulate the modulation of IPSC charge, supporting the hypothesis that presynaptic loading of glycine negatively impact the GABAergic transmission in mixed interneurons through a competition for vesicular filling. Our results suggest that heterogeneity of neurotransmitter supply within the GoC class may provide a presynaptic mechanism to tune the gain of the stereotypic granular layer microcircuit, thereby expanding the realm of possible dynamic behavior.

neuroscience↗

bioRxiv2018

Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

Imaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed high-resolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.

neuroscience↗

bioRxiv2018

Voltage- and Branch-specific Climbing Fiber Responses in Purkinje Cells

Climbing fibers (CFs) provide instructive signals driving cerebellar learning. However, conflicting experimental studies have been reported about the reliability of CF mediated Ca2+ influx in Purkinje cell (PC) distal dendrites. Mechanisms causing the wide variation in duration and spikelet numbers of complex spikes (CSs) have not been explored systematically. Using a new experimentally validated PC model, we describe the full range of modifiability of CF responses to explain the experimental data and make new predictions. We find voltage state gates the initiation and propagation of dendritic spikes. PC dendrites exhibit inhomogeneous excitability with individual branches as computational units for CF input. Somatic CSs are regulated by voltage state, CF activation phase and instantaneous CF firing rate. Concurrent synaptic inputs can affect CSs by modulating dendritic responses in a spatially precise way. These voltage- and branch-specific CF responses will increase dendritic computational capacity and give PCs an active role in integrating CF signals.

neuroscience↗