Biology subjects

Villette, V.

Publications and source records attributed to Villette, V..

10 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↗

bioRxiv2026

A red-emitting, genetically encoded indicator for two-photon voltage recording in vivo

Genetically encoded voltage indicators (GEVIs) enable minimally invasive, cell-type-specific optical measurements of neuronal membrane potential with millisecond temporal resolution. Red-shifted GEVIs are especially advantageous because they permit spectral multiplexing with complementary sensors and enable all-optical circuit interrogation in combination with blue-light-activated opsins. Despite these advantages, existing red GEVIs remain poorly suited for in vivo use due to limited performance under two-photon (2P) excitation, the predominant modality for deep-tissue imaging. Here, we introduce VADER1, a red GEVI that overcomes this limitation and enables reliable spike detection in vivo under 2P illumination. Under 2P excitation, VADER1 supports extended voltage imaging with both random-access and resonant-scanning microscopy, enables recordings from neurons as deep as cortical layer 5, and allows dual-color imaging with calcium indicators. By filling a critical spectral gap, VADER1 enables integrated optical measurements of fast electrical activity alongside other neural signals and establishes a foundation for two-photon all-optical electrophysiology.

neuroscience↗

bioRxiv2026

Designer indicators for two-photon recording of subthreshold voltage dynamics

Subthreshold voltage dynamics are critical for neuronal information integration, yet they remain understudied in vivo due to the limitations of current tools. While genetically encoded voltage indicators (GEVIs) offer a promising alternative, their application for deep-tissue recording using two-photon (2P) microscopy--a preferred method for deep-tissue recording--has been hindered by insufficient sensitivity for detecting millivolt-scale subthreshold signals. Here, we refined our multiparametric two-photon high-throughput screening platform to develop two novel GEVIs, JEDI3sub and JEDI3hyp, tailored explicitly for subthreshold voltage detection. Through fast 2P optical recording in awake, behaving mice, we demonstrated the superior sensitivity of JEDI3 indicators compared to JEDI-2P. We also showed that JEDI3sub can track population-level subthreshold optical tuning, while JEDI3hyp reliably captured subthreshold dynamics associated with sharp-wave ripple oscillations in hippocampal PV interneurons. Finally, JEDI3hyp facilitated extended imaging of brain-state-dependent, millivolt-scale subthreshold voltage changes across deep-layer somas, fine dendritic structures, and diverse cell types. By addressing the critical gap in 2P optical recording of subthreshold voltage dynamics, JEDI3 indicators open new avenues for studying neural information processing and its alterations in health and disease.

neuroscience↗

bioRxiv2026

A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo

Genetically encoded voltage indicators (GEVIs) enable cell-type-specific optical readout of membrane potential, but two-photon (2P) spike detection has been hampered by low signal-to-noise and ultrafast off-kinetics, restricting use to specialized microscopes. We introduce FORCE1s, a green, positive-going GEVI engineered to make robust 2P voltage imaging broadly accessible. FORCE1s brightens from a dark baseline during depolarization, reports spikes with [~]100% {Delta}F/F in awake mice, and displays repolarization kinetics that are tuned for reliable spike detection at sub-kilohertz frame rates. As a result, FORCE1s supports spike-resolved multi-cell recordings on standard resonant-scanning microscopes, and further scales to larger fields of view and neuron counts on advanced modalities. FORCE1s also enables multiplexed voltage-neurotransmitter imaging and extended recordings in freely moving mice using a compact, affordable MEMS-based 2P miniscope. Together, these advances establish FORCE1s as a community-ready tool that democratizes deep-tissue voltage imaging across platforms and experimental contexts.

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↗

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↗

bioRxiv2018

Internal representation of hippocampal neuronal population span a time-distance continuum

The hippocampus plays a critical role in episodic memory: the sequential representation of visited places and experienced events. This function is mirrored by hippocampal activity that self organizes into sequences of neuronal activation that integrate spatio-temporal information. What are the underlying mechanisms of such integration is still unknown. Single cell activity was recently shown to combine time and distance information; however, it remains unknown whether a degree of tuning between space and time can be defined at the network level. Here, combining daily calcium imaging of CA1 sequence dynamics in running head-fixed mice and network modeling, we show that CA1 network activity tends to represent a specific combination of space and time at any given moment, and that the degree of tuning can shift within a continuum from one day to the next. Our computational model shows that this shift in tuning can happen under the control of the external drive power. We propose that extrinsic global inputs shape the nature of spatio-temporal integration in the hippocampus at the population level depending on the task at hand, a hypothesis which may guide future experimental studies.\n\nSignificance StatementThe hippocampus organizes experience in sequences of events that form episodic memory. How are time and space internally computed in the hippocampus in the absence of sequential external inputs? Here we show that time and space are integrated together within the hippocampal network with different degrees of tuning across days. This was found by recording the activity of hundreds of pyramidal cells for several days. We also propose a mechanism supporting such spatio-temporal integration based on a ring attractor network model: the degree of tuning between space and time can be adjusted by modulating the power of a non-sequential external excitatory drive. In this way, the hippocampus is able to generate a spatio-temporal representation tuned to the task at hand.

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

Synaptic properties and network state-dependent recruitment of VIP-expressing interneuron-specific interneurons in the CA1 hippocampus

In the hippocampus, a highly specialized population of vasoactive intestinal peptide (VIP)-expressing interneuron-specific (IS) inhibitory cells provides local circuit disinhibition via preferential innervation of different types of GABAergic interneurons. While disinhibition can be critical in modulating network activity and different forms of hippocampal learning, the synaptic and integrative properties of IS cells and their recruitment during network oscillations remain unknown. Using a combination of patch-clamp recordings, photostimulation, computational modelling as well as recordings of network oscillations simultaneously with two-photon Ca2+-imaging in awake mice in vivo, we identified synaptic mechanisms that can control the firing of IS cells, and explored their impact on the cell recruitment during theta oscillations and sharp-wave-associated ripples. We found that IS cells fire spikes in response to both the Schaffer collateral and the temporoammonic pathway activation. Moreover, integrating their intrinsic and synaptic properties into computational models predicted recruitment of these cells during the rising to peak phases of theta oscillations and during ripples depending on inhibitory contributions. In vivo Ca2+-imaging in awake mice confirmed in part the theoretical predictions, revealing a significant speed modulation of IS cells and their preferential albeit delayed recruitment during theta-run epochs, with firing at the rising phase to peak of the theta cycle. However, it also uncovered that IS cells are not activated during ripples. Thus, given the preferential theta-modulated firing of IS cells in awake hippocampus, we postulate that these cells may be important for information gating during spatial navigation and memory encoding.

neuroscience↗

bioRxiv2018

Connectivity and network state-dependent recruitment of long-range VIP-GABAergic neurons in the mouse hippocampus

GABAergic interneurons in the hippocampus provide for local and long-distance coordination of neurons in functionally connected areas. Vasoactive intestinal peptide-expressing (VIP+) interneurons occupy a distinct niche in circuitry as many of them specialize in innervating GABAergic cells, thus providing network disinhibition. In the CA1 hippocampus, VIP+ interneuron-selective cells target local interneurons. Here, we discovered a novel type of VIP+ neuron whose axon innervates CA1 and also projects to the subiculum (VIP-LRPs). VIP-LRPs showed specific molecular properties and targeted interneurons within the CA1 area but both interneurons and pyramidal cells within subiculum. They were interconnected through gap junctions but demonstrated sparse spike coupling in vitro. In awake mice, VIP-LRPs decreased their activity during theta-run epochs and were more active during quiet wakefulness but not coupled to sharp-wave ripples. Together, the data provide new evidence for VIP interneuron molecular diversity and functional specialization in controlling cell ensembles along the hippocampo-subicular axis.

neuroscience↗