A flexible two-photon fiberscope for fast activity imaging and precise optogenetic photostimulation of neurons in freely moving mice.

We developed a flexible two-photon microendoscope (2P-FENDO) capable of all-optical brain investigation at near cellular resolution in freely moving mice. The system performs fast two-photon (2P) functional imaging and 2P holographic photostimulation of single and multiple cells using axially confined extended spots. Proof-of-principle experiments were performed in freely moving mice co-expressing jGCaMP7s and the opsin ChRmine in the visual or barrel cortex.

All-optical inter-layers functional connectivity investigation in the mouse retina.

We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution.

Single-Cell Resolution Optogenetics Via Expression of Soma-Targeted Rhodopsins.

Optogenetics allows control of neural activity in genetically targeted neuron populations by light. Optogenetic control of individual neurons in neural circuits would enable powerful, causal investigations of neural connectivity and function at single-cell level and provide insights into how neural circuits operate. Such single-cell resolution optogenetics in neuron populations requires precise sculpting of light and subcellular targeting of optogenetic molecules.

Optimized Chronos sets the clock for optogenetic hearing restoration.

The coming of age of optogenetics has motivated the development of clinical applications. Improved hearing restoration by optical cochlear implants is one such promising development. However, slow closing of light‐gated ion channels has remained an obstacle for achieving the high temporal fidelity required for optogenetic coding.

Publisher Correction: Temporally precise single-cell-resolution optogenetics.

In the supplementary information originally posted online, Supplementary Tables 1-5 and the Supplementary Note were missing. The error has been corrected online.

Temporally precise single-cell-resolution optogenetics.

Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons.

A novel multisite confocal system for rapid Ca imaging from submicron structures in brain slices.

In brain slices, resolving fast Ca fluorescence signals from submicron structures is typically achieved using 2-photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high-resolution CMOS camera.

Submillisecond Optogenetic Control of Neuronal Firing with Two-Photon Holographic Photoactivation of Chronos.

Optogenetic neuronal network manipulation promises to unravel a long-standing mystery in neuroscience: how does microcircuit activity relate causally to behavioral and pathological states? The challenge to evoke spikes with high spatial and temporal complexity necessitates further joint development of light-delivery approaches and custom opsins. Two-photon (2P) light-targeting strategies demonstrated in-depth generation of action potentials in photosensitive neurons both and , but thus far lack the temporal precision necessary to induce precisely timed spiking events. Here, we show that efficient current integration enabled by 2P holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice.

Imaging membrane potential changes from dendritic spines using computer-generated holography.

Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor electrical subthreshold events as they travel from synapses on distal dendrites and summate at particular locations to initiate action potentials. It is now possible to carry out these measurements at the scale of individual dendritic spines using voltage imaging.

Mechanisms and functional roles of glutamatergic synapse diversity in a cerebellar circuit.

Synaptic currents display a large degree of heterogeneity of their temporal characteristics, but the functional role of such heterogeneities remains unknown. We investigated in rat cerebellar slices synaptic currents in Unipolar Brush Cells (UBCs), which generate intrinsic mossy fibers relaying vestibular inputs to the cerebellar cortex. We show that UBCs respond to sinusoidal modulations of their sensory input with heterogeneous amplitudes and phase shifts.

Distinct roles of Eps8 in the maturation of cochlear and vestibular hair cells.

Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling, has been shown to cause deafness in mice.

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures.

Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity.

Fine Tuning of CaV1.3 Ca2+ channel properties in adult inner hair cells positioned in the most sensitive region of the Gerbil Cochlea.

Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca(2+) inflow through Ca(V)1.3 (L-type) Ca(2+) channels.

Burst activity and ultrafast activation kinetics of CaV1.3 Ca²⁺ channels support presynaptic activity in adult gerbil hair cell ribbon synapses.

Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca²⁺ entry through CaV1.3 Ca²⁺ channels. Despite the crucial role of CaV1.

Position-dependent patterning of spontaneous action potentials in immature cochlear inner hair cells.

Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning.

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells.

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice.

Elementary properties of CaV1.3 Ca(2+) channels expressed in mouse cochlear inner hair cells.

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca(V)1.

Calyx and dimorphic neurons of mouse Scarpa's ganglion express histamine H3 receptors.

Background: Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. The site of action of these drugs however has not been elucidated yet. Recent works on amphibians showed that histamine H3 receptor antagonists, e.

Histamine H1 receptors are expressed in mouse and frog semicircular canal sensory epithelia.

Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. Their site and mechanism of action, however, are still poorly understood. To increase our knowledge of the histaminergic system in the vestibular organs, we have investigated the expression of H1 and H3 histamine receptors in the frog and mouse semicircular canal sensory epithelia.

Single-channel L-type Ca2+ currents in chicken embryo semicircular canal type I and type II hair cells.

Few data are available concerning single Ca channel properties in inner ear hair cells and particularly none in vestibular type I hair cells. By using the cell-attached configuration of the patch-clamp technique in combination with the semicircular canal crista slice preparation, we determined the elementary properties of voltage-dependent Ca channels in chicken embryo type I and type II hair cells. The pipette solutions included Bay K 8644.

Ca(2+) currents and voltage responses in Type I and Type II hair cells of the chick embryo semicircular canal.

Type I and Type II hair cells, and Type II hair cells located in different zones of the semicircular canal crista, express different patterns of voltage-dependent K channels, each one specifically shaping the hair cell receptor potential. We report here that, close to hatching, chicken embryo semicircular canal Type I and Type II hair cells express a similar voltage-dependent L-type calcium current (I(Ca)), whose main features are: activation above -60 mV, fast activation kinetics, and scarce inactivation. I(Ca) should be already active at rest in Zone 1 Type II hair cells, whose resting membrane potential was on average slightly less negative than -60 mV.