Benjamin Mathieu

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

Activity-Dependent Gating of Calcium Spikes by A-type K+ Channels Controls Climbing Fiber Signaling in Purkinje Cell Dendrites

Summary In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.

Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum

The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly. Summary: Time-lapse imaging, cell ablation and MSD plot analysis reveal relative random, directed or confined displacement of differentiating epithelial cells during taste bud development in zebrafish.

Active intermixing of indirect and direct neurons builds the striatal mosaic

The striatum controls behaviors via the activity of direct and indirect pathway projection neurons (dSPN and iSPN) that are intermingled in all compartments. While such mosaic ensures the balanced activity of the two pathways, how it emerges remains largely unknown. Here, we show that both SPN populations are specified embryonically and progressively intermix through multidirectional iSPN migration. Using conditional mutants of the dSPN-specific transcription factor Ebf1, we found that inactivating this gene impaired selective dSPN properties, including axon pathfinding, whereas molecular and functional features of iSPN were preserved. Remarkably, Ebf1 mutation disrupted iSPN/dSPN intermixing, resulting in an uneven distribution. Such architectural defect was selective of the matrix compartment, revealing that intermixing is a parallel process to compartment formation. Our study reveals that, while iSPN/dSPN specification is largely independent, their intermingling emerges from an active migration of iSPN, thereby providing a novel framework for the building of striatal architecture.

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.

Optogenetic stimulation of complex spatio-temporal activity patterns by acousto-optic light steering probes cerebellar granular layer integrative properties

Optogenetics provides tools to control afferent activity in brain microcircuits. However, this requires optical methods that can evoke asynchronous and coordinated activity within neuronal ensembles in a spatio-temporally precise way. Here we describe a light patterning method, which combines MHz acousto-optic beam steering and adjustable low numerical aperture Gaussian beams, to achieve fast 2D targeting in scattering tissue. Using mossy fiber afferents to the cerebellar cortex as a testbed, we demonstrate single fiber optogenetic stimulation with micron-scale lateral resolution, >100 µm depth-penetration and 0.1 ms spiking precision. Protracted spatio-temporal patterns of light delivered by our illumination system evoked sustained asynchronous mossy fiber activity with excellent repeatability. Combining optical and electrical stimulations, we show that the cerebellar granular layer performs nonlinear integration, whereby sustained mossy fiber activity provides a permissive context for the transmission of salient inputs, enriching combinatorial views on mossy fiber pattern separation.

Fast optimization wavefront shaping with acousto-optic deflectors (Conference Presentation)

Since a decade, wavefront shaping techniques has allowed to coherently manipulate speckle patterns. It opens the possibility to focus light through complex media and ultimately to image in them, provided that the medium can be considered as stationary during the process. However, scattering by tissues evolves over millisecond timescales, creating a fast decorrelation of the speckle pattern, thus limiting the use of this technique for in vivo microscopy. Therefore, focusing through biological tissues requires fast wavefront shaping devices, sensors and algorithms. It has been demonstrated by Akemann et al that an Acousto-Optic Deflector (AOD) time locked on the output laser pulses of a regenerative amplifier can be used as an arbitrary 1D beam shaper: the locally modulated acousto-optic phase grating allows the spatial control of the laser pulse wavefront, with refresh rate of several tens up to several hundreds of kHz, limited by the size of the AOD aperture. We have investigated through simulations and experiments, the use of two crossed AODs to implement 2D spatial wavefront shaping, and perform focusing by optimization through a scattering media. We have used different algorithms adapted to this grating modulator and analyzed in each case the AOD bandwidth used, the speed of convergence and the maximum intensity enhancement. In particular, we have shown that two crossed 1D modulators provide larger enhancement than a single 2D wavefront shaper with the same number of pixels. We will present our latest results towards achieving the ultimate optimization, limited by the AOD speed of 40 kHz.