Seeing our hand or a tool during visually-guided actions: Different effects on the somatosensory and visual cortices.

The processing of proprioceptive information in the context of a conflict between visual and somatosensory feedbacks deteriorates motor performance. Previous studies have shown that seeing one's hand increases the weighting assigned to arm somatosensory inputs. In this light, we hypothesized that the sensory conflict, when tracing the contour of a shape with mirror-reversed vision, will be greater for participants who trace with a stylus seen in their hand (Hand group, n = 17) than for participants who trace with the tip of rod without seen their hand (Tool group, n = 15). Based on this hypothesis, we predicted that the tracing performance with mirror vision will be more deteriorated for the Hand group than for the Tool group, and we predicted a greater gating of somatosensory information for the Hand group to reduce the sensory conflict. The participants of both groups followed the outline of a shape in two visual conditions. Direct vision: the participants saw the hand or portion of a light 40 cm rod directly. Mirror Vision: the hand or the rod was seen through a mirror. We measured tracing performance using a digitizing tablet and the cortical activity with electroencephalography. Behavioral analyses revealed that the tracing performance of both groups was similarly impaired by mirror vision. However, contrasting the spectral content of the cortical oscillatory activity between the Mirror and Direct conditions, we observed that tracing with mirror vision resulted in significantly larger alpha (8-12 Hz) and beta (15-25 Hz) powers in the somatosensory cortex for participants of the Hand group. The somatosensory alpha and beta powers did not significantly differ between Mirror and Direct vision conditions for the Tool group. For both groups, tracing with mirror vision altered the activity of the visual cortex: decreased alpha power for the Hand group, decreased alpha and beta power for the Tool group. Overall, these results suggest that seeing the hand enhanced the sensory conflict when tracing with mirror vision and that the increase of alpha and beta powers in the somatosensory cortex served to reduce the weight assigned to somatosensory information. The increased activity of the visual cortex observed for both groups in the mirror vision condition suggests greater visual processing with increased task difficulty. Finally, the fact that the participants of the Tool group did not show better tracing performance than those of the Hand group suggests that tracing deterioration resulted from a sensorimotor conflict (as opposed to a visuo-proprioceptive conflict).

Keeping in touch with our hidden side.

Previous studies have shown that the sensory modality used to identify regions of the body hidden from sight, but frequently viewed, influences the type of the body representation employed for reaching them with the finger. The question then arises as to whether this observation also applies to body regions which are rarely, if ever, viewed. We used an established technique for pinpointing the type of body representation used for the spatial encoding of targets which consisted of assessing the effect of peripheral gaze fixation on the pointing accuracy. More precisely, an exteroceptive, visually dependent, body representation is thought to be used if gaze deviation induces a deviation of the pointing movement. Three light-emitting diodes (LEDs) were positioned at the participants' eye level at -25 deg, 0 deg and +25 deg. Without moving the head, the participant fixated the lit LED before the experimenter indicated one of the three target head positions: topmost point of the head (vertex) and two other points located at the front and back of the head. These targets were either verbal-cued or tactile-cued and the participants had to reach them with their index finger. We analysed the accuracy of the movements directed to the topmost point of the head, which is a well-defined, yet out of view anatomical point. Based on the possibility of the brain to create visual representations of the body areas that remain out of view, we hypothesized that the position of the vertex is encoded using an exteroceptive body representation, both when verbally or tactile-cued. Results revealed that the pointing errors were biased in the opposite direction of gaze fixation for both verbal-cued and tactile-cued targets, suggesting the use of a vision-dependent exteroceptive body representation. The enhancement of the visual body representations by sensorimotor processes was suggested by the greater pointing accuracy when the vertex was identified by tactile stimulation compared to verbal instruction. Moreover, a control condition showed that participants were more accurate in indicating the position of their own vertex than the vertex of other people. Together, our results suggest that the position of rarely viewed body parts are spatially encoded by an exteroceptive body representation and that non-visual sensorimotor processes are involved in the constructing of this representation.

Fast optical recording of neuronal activity by three-dimensional custom-access serial holography.

Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

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 cellular mosaic ensures the balanced activity of the two pathways, its developmental origin and pattern remains largely unknown. Here, we show that both SPN populations are specified embryonically and intermix progressively through multidirectional iSPN migration. Using conditional mutant mice, we found that inactivation of the dSPN-specific transcription factor Ebf1 impairs selective dSPN properties, including axon pathfinding, while molecular and functional features of iSPN were preserved. Ebf1 mutation disrupted iSPN/dSPN intermixing, resulting in an uneven distribution. Such architectural defect was selective of the matrix compartment, highlighting that intermixing is a parallel process to compartment formation. Our study reveals 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.

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.

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.

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.

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.

Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in Purkinje cell dendrites.

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.

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.