Fast 2-photon stimulation using holographic patterns.

Two decades after its introduction, optogenetics - a biological technique to control the activity of neurons or other cell types with light - remains a cutting edge and promising tool to study biological processes. Its increasing usage in research varies widely from causally exploring biological mechanisms and neural computations, to neurostimulation and sensory restauration. To stimulate neurons in the brain, a variety of approaches have been developed to generate precise spatiotemporal light patterns. Yet certain constrains still exists in the current optical techniques to activate a neuronal population with both cellular resolution and millisecond precision. Here, we describe an experimental setup allowing to stimulate a few tens of neurons in a plane at sub-millisecond rates using 2-photon activation. A liquid crystal on silicon spatial light modulator (LCoS-SLM) was used to generate spatial patterns in 2 dimensions. The image of the patterns was formed on the plane of a digital micromirror device (DMD) that was used as a fast temporal modulator of each region of interest. Using fluorescent microscopy and patch-clamp recording of neurons in culture expressing the light-gated ion channels, we characterized the temporal and spatial resolution of the microscope. We described the advantages of combining the LCoS-SLM with the DMD to maximize the temporal precision, modulate the illumination amplitude, and reduce background activation. Finally, we showed that this approach can be extended to patterns in 3 dimensions. We concluded that the methodology is well suited to address important questions about the role of temporal information in neuronal coding.

Signatures of cochlear processing in neuronal coding of auditory information.

The vertebrate ear is endowed with remarkable perceptual capabilities. The faintest sounds produce vibrations of magnitudes comparable to those generated by thermal noise and can nonetheless be detected through efficient amplification of small acoustic stimuli. Two mechanisms have been proposed to underlie such sound amplification in the mammalian cochlea: somatic electromotility and active hair-bundle motility. These biomechanical mechanisms may work in concert to tune auditory sensitivity. In addition to amplitude sensitivity, the hearing system shows exceptional frequency discrimination allowing mammals to distinguish complex sounds with great accuracy. For instance, although the wide hearing range of humans encompasses frequencies from 20 Hz to 20 kHz, our frequency resolution extends to one-thirtieth of the interval between successive keys on a piano. In this article, we review the different cochlear mechanisms underlying sound encoding in the auditory system, with a particular focus on the frequency decomposition of sounds. The relation between peak frequency of activation and location along the cochlea - known as tonotopy - arises from multiple gradients in biophysical properties of the sensory epithelium. Tonotopic mapping represents a major organizational principle both in the peripheral hearing system and in higher processing levels and permits the spectral decomposition of complex tones. The ribbon synapses connecting sensory hair cells to auditory afferents and the downstream spiral ganglion neurons are also tuned to process periodic stimuli according to their preferred frequency. Though sensory hair cells and neurons necessarily filter signals beyond a few kHz, many animals can hear well beyond this range. We finally describe how the cochlear structure shapes the neural code for further processing in order to send meaningful information to the brain. Both the phase-locked response of auditory nerve fibers and tonotopy are key to decode sound frequency information and place specific constraints on the downstream neuronal network.

Friction from Transduction Channels' Gating Affects Spontaneous Hair-Bundle Oscillations.

Hair cells of the inner ear can power spontaneous oscillations of their mechanosensory hair bundle, resulting in amplification of weak inputs near the characteristic frequency of oscillation. Recently, dynamic force measurements have revealed that delayed gating of the mechanosensitive ion channels responsible for mechanoelectrical transduction produces a friction force on the hair bundle. The significance of this intrinsic source of dissipation for the dynamical process underlying active hair-bundle motility has remained elusive. The aim of this work is to determine the role of friction in spontaneous hair-bundle oscillations. To this end, we characterized key oscillation properties over a large ensemble of individual hair cells and measured how viscosity of the endolymph that bathes the hair bundles affects these properties. We found that hair-bundle movements were too slow to be impeded by viscous drag only. Moreover, the oscillation frequency was only marginally affected by increasing endolymph viscosity by up to 30-fold. Stochastic simulations could capture the observed behaviors by adding a contribution to friction that was 3-8-fold larger than viscous drag. The extra friction could be attributed to delayed changes in tip-link tension as the result of the finite activation kinetics of the transduction channels. We exploited our analysis of hair-bundle dynamics to infer the channel activation time, which was ∼1 ms. This timescale was two orders-of-magnitude shorter than the oscillation period. However, because the channel activation time was significantly longer than the timescale of mechanical relaxation of the hair bundle, channel kinetics affected hair-bundle dynamics. Our results suggest that friction from channel gating affects the waveform of oscillation and that the channel activation time can tune the characteristic frequency of the hair cell. We conclude that the kinetics of transduction channels' gating plays a fundamental role in the dynamic process that shapes spontaneous hair-bundle oscillations.

Transduction channels' gating can control friction on vibrating hair-cell bundles in the ear.

Hearing starts when sound-evoked mechanical vibrations of the hair-cell bundle activate mechanosensitive ion channels, giving birth to an electrical signal. As for any mechanical system, friction impedes movements of the hair bundle and thus constrains the sensitivity and frequency selectivity of auditory transduction. Friction is generally thought to result mainly from viscous drag by the surrounding fluid. We demonstrate here that the opening and closing of the transduction channels produce internal frictional forces that can dominate viscous drag on the micrometer-sized hair bundle. We characterized friction by analyzing hysteresis in the force-displacement relation of single hair-cell bundles in response to periodic triangular stimuli. For bundle velocities high enough to outrun adaptation, we found that frictional forces were maximal within the narrow region of deflections that elicited significant channel gating, plummeted upon application of a channel blocker, and displayed a sublinear growth for increasing bundle velocity. At low velocity, the slope of the relation between the frictional force and velocity was nearly fivefold larger than the hydrodynamic friction coefficient that was measured when the transduction machinery was decoupled from bundle motion by severing tip links. A theoretical analysis reveals that channel friction arises from coupling the dynamics of the conformational change associated with channel gating to tip-link tension. Varying channel properties affects friction, with faster channels producing smaller friction. We propose that this intrinsic source of friction may contribute to the process that sets the hair cell's characteristic frequency of responsiveness.

Phantom tones and suppressive masking by active nonlinear oscillation of the hair-cell bundle.

Processing of two-tone stimuli by the auditory system introduces prominent masking of one frequency component by the other as well as additional "phantom" tones that are absent in the sound input. Mechanical correlates of these psychophysical phenomena have been observed in sound-evoked mechanical vibrations of the mammalian cochlea and are thought to originate in sensory hair cells from the intrinsic nonlinearity associated with mechano-electrical transduction by ion channels. However, nonlinearity of the transducer is not sufficient to explain the rich phenomenology of two-tone interferences in hearing. Here we show that active oscillatory movements of single hair-cell bundles elicit two-tone suppression and distortions that are shaped by nonlinear amplification of periodic stimuli near the characteristic frequency of spontaneous oscillations. When both stimulus frequencies enter the bandwidth of the hair-bundle amplifier, two-tone interferences display level functions that are characteristic both of human psychoacoustics and of in vivo mechanical measurements in auditory organs. Our work distinguishes the frequency-dependent nonlinearity that emerges from the active process that drives the hair bundle into spontaneous oscillations from the passive nonlinear compliance associated with the direct gating of transduction channels by mechanical force. Numerical simulations based on a generic description of an active dynamical system poised near an oscillatory instability--a Hopf bifurcation--account quantitatively for our experimental observations. In return, we conclude that the properties of two-tone interferences in hearing betray the workings of self-sustained "critical" oscillators, which function as nonlinear amplifying elements in the inner ear.

Coupling a sensory hair-cell bundle to cyber clones enhances nonlinear amplification.

The vertebrate ear benefits from nonlinear mechanical amplification to operate over a vast range of sound intensities. The amplificatory process is thought to emerge from active force production by sensory hair cells. The mechano-sensory hair bundle that protrudes from the apical surface of each hair cell can oscillate spontaneously and function as a frequency-selective, nonlinear amplifier. Intrinsic fluctuations, however, jostle the response of a single hair bundle to weak stimuli and seriously limit amplification. Most hair bundles are mechanically coupled by overlying gelatinous structures. Here, we assayed the effects of mechanical coupling on the hair-bundle amplifier by combining dynamic force clamp of a hair bundle from the bullfrog's saccule with real-time stochastic simulations of hair-bundle mechanics. This setup couples the hair bundle to two virtual hair bundles, called cyber clones, and mimics a situation in which the hair bundle is elastically linked to two neighbors with similar characteristics. We found that coupling increased the coherence of spontaneous hair-bundle oscillations. By effectively reducing noise, the synergic interplay between the hair bundle and its cyber clones also enhanced amplification of sinusoidal stimuli. All observed effects of coupling were in quantitative agreement with simulations. We argue that the auditory amplifier relies on hair-bundle cooperation to overcome intrinsic noise limitations and achieve high sensitivity and sharp frequency selectivity.

Propagation of temporal and rate signals in cultured multilayer networks.

Analyses of idealized feedforward networks suggest that several conditions have to be satisfied in order for activity to propagate faithfully across layers. Verifying these concepts experimentally has been difficult owing to the vast number of variables that must be controlled. Here, we cultured cortical neurons in a chamber with sequentially connected compartments, optogenetically stimulated individual neurons in the first layer with high spatiotemporal resolution, and then monitored the subthreshold and suprathreshold potentials in subsequent layers. Brief stimuli delivered to the first layer evoked a short-latency transient response followed by sustained activity. Rate signals, carried by the sustained component, propagated reliably through 4 layers, unlike idealized feedforward networks, which tended strongly towards synchrony. Moreover, temporal jitter in the stimulus was transformed into a rate code and transmitted to the last layer. This novel mode of propagation occurred in the balanced excitatory-inhibitory regime and is mediated by NMDA-mediated receptors and recurrent activity.

Molecular characterization of the ankle-link complex in cochlear hair cells and its role in the hair bundle functioning.

Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1-/- mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1-/- mice. In postnatal day 7 Vlgr1-/- mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-link-mediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1-/- mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1-/- mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.

Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control.

We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital Light Processing (DLP) projector. The setup is able to stimulate tens to hundreds neurons with independent trains of light pulses that evoked action potentials with high temporal resolution. During photostimulation, network activity was monitored using patch-clamp recordings of up to 4 neurons. The experiment is ideally suited to study recurrent network dynamics or biological processes such as plasticity or homeostasis in a network of neurons when a sub-population is activated by distinct stimuli whose characteristics (correlation, rate, and, size) were finely controlled.

Synaptic scaling rule preserves excitatory-inhibitory balance and salient neuronal network dynamics.

The balance between excitation and inhibition (E-I balance) is maintained across brain regions though the network size, strength and number of synaptic connections, and connection architecture may vary substantially. We use a culture preparation to examine the homeostatic synaptic scaling rules that produce E-I balance and in vivo-like activity. We show that synaptic strength scales with the number of connections K as ∼ , close to the ideal theoretical value. Using optogenetic techniques, we delivered spatiotemporally patterned stimuli to neurons and confirmed key theoretical predictions: E-I balance is maintained, active decorrelation occurs and the spiking correlation increases with firing rate. Moreover, the trial-to-trial response variability decreased during stimulation, as observed in vivo. These results-obtained in generic cultures, predicted by theory and observed in the intact brain-suggest that the synaptic scaling rule and resultant dynamics are emergent properties of networks in general.

Artificial rheotaxis.

Motility is a basic feature of living microorganisms, and how it works is often determined by environmental cues. Recent efforts have focused on developing artificial systems that can mimic microorganisms, in particular their self-propulsion. We report on the design and characterization of synthetic self-propelled particles that migrate upstream, known as positive rheotaxis. This phenomenon results from a purely physical mechanism involving the interplay between the polarity of the particles and their alignment by a viscous torque. We show quantitative agreement between experimental data and a simple model of an overdamped Brownian pendulum. The model notably predicts the existence of a stagnation point in a diverging flow. We take advantage of this property to demonstrate that our active particles can sense and predictably organize in an imposed flow. Our colloidal system represents an important step toward the realization of biomimetic microsystems with the ability to sense and respond to environmental changes.

The physical basis of active mechanosensitivity by the hair-cell bundle.

PURPOSE OF REVIEW: Hearing starts with the deflection of the hair bundle that sits on top of each mechanosensory hair cell. Recent advances indicate that the hair bundle mechanically amplifies its inputs to participate in the active process that boosts the ear's technical specifications. This review integrates experimental and modeling studies to dissect the mechanisms of active mechanosensation by the hair-cell bundle. RECENT FINDINGS: The exquisite mechanosensitivity of the hair-cell bundle results from a precisely choreographed interplay between a structure of mechanically coupled stereocilia that ensures efficient transmission of sound-energy to the transduction machinery, Ca-driven adaptation that provides fast electromechanical feedback on hair-bundle movements, and a mechanical nonlinearity inherent to the transduction process that fosters autonomous hair-bundle oscillations. In cochlear outer hair cells, cooperation between active hair-bundle motility and somatic electromotility brings the cochlear partition to the brink of an oscillatory instability, at which general physical laws ensure optimal properties for auditory detection. SUMMARY: The study of active hair-bundle mechanics promotes a general principle for auditory detection that is based on the generic properties of self-sustained mechanical oscillators. This principle may guide future engineering design of cochlear implants.