Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks.

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

Neurons differentiate magnitude and location of mechanical stimuli.

Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.

Restoration of high-sensitivity and adapting vision with a cone opsin.

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision.

Author Correction: Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Kevin J. Cao and Richard H. Kramer, who developed extended release with beta cyclodextrin, were inadvertently omitted from the author list and author contributions section of this Article. These errors have now been corrected in both the PDF and HTML versions of the Article.

Optogenetic Retinal Gene Therapy with the Light Gated GPCR Vertebrate Rhodopsin.

In retinal disease, despite the loss of light sensitivity as photoreceptors die, many retinal interneurons survive in a physiologically and metabolically functional state for long periods. This provides an opportunity for treatment by genetically adding a light sensitive function to these cells. Optogenetic therapies are in development, but, to date, they have suffered from low light sensitivity and narrow dynamic response range of microbial opsins. Expression of light-sensitive G protein coupled receptors (GPCRs), such as vertebrate rhodopsin , can increase sensitivity by signal amplification , as shown by several groups. Here, we describe the methods to (1) express light gated GPCRs in retinal neurons, (2) record light responses in retinal explants in vitro, (3) record cortical light responses in vivo, and (4) test visually guided behavior in treated mice.

Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 ("SNAG-mGluR2") evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics.

Inertial picobalance reveals fast mass fluctuations in mammalian cells.

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a 'picobalance'), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

Mechanical Stimulation of Piezo1 Receptors Depends on Extracellular Matrix Proteins and Directionality of Force.

Piezo receptors convert mechanical forces into electrical signals. In mammals, they play important roles in basic physiological functions including proprioception, sensation of touch, and vascular development. However, basic receptor properties like the gating mechanism, the interaction with extracellular matrix (ECM) proteins, and the response to mechanical stimulation, remain poorly understood. Here, we establish an atomic force microscopy (AFM)-based assay to mechanically stimulate Piezo1 receptors in living animal cells, while monitoring receptor activation in real-time using functional calcium imaging. Our experiments show that in the absence of ECM proteins Piezo1 receptors are relatively insensitive to mechanical forces pushing the cellular membrane, whereas they can hardly be activated by mechanically pulling the membrane. Yet, if conjugated with Matrigel, a mix of ECM proteins, the receptors become sensitized. Thereby, forces pulling the cellular membrane activate the receptor much more efficiently compared to pushing forces. Finally, we found that collagen IV, a component of the basal lamina, which forms a cohesive network and mechanical connection between cells, sensitizes Piezo1 receptors to mechanical pulling.

Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity.

Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.

A Device for Human Ultrasonic Echolocation.

OBJECTIVE: We present a device that combines principles of ultrasonic echolocation and spatial hearing to provide human users with environmental cues that are 1) not otherwise available to the human auditory system, and 2) richer in object and spatial information than the more heavily processed sonar cues of other assistive devices. The device consists of a wearable headset with an ultrasonic emitter and stereo microphones with affixed artificial pinnae. The goal of this study is to describe the device and evaluate the utility of the echoic information it provides. METHODS: The echoes of ultrasonic pulses were recorded and time stretched to lower their frequencies into the human auditory range, then played back to the user. We tested performance among naive and experienced sighted volunteers using a set of localization experiments, in which the locations of echo-reflective surfaces were judged using these time-stretched echoes. RESULTS: Naive subjects were able to make laterality and distance judgments, suggesting that the echoes provide innately useful information without prior training. Naive subjects were generally unable to make elevation judgments from recorded echoes. However, trained subjects demonstrated an ability to judge elevation as well. CONCLUSION: This suggests that the device can be used effectively to examine the environment and that the human auditory system can rapidly adapt to these artificial echolocation cues. SIGNIFICANCE: Interpreting and interacting with the external world constitutes a major challenge for persons who are blind or visually impaired. This device has the potential to aid blind people in interacting with their environment.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells.

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

Optical control of metabotropic glutamate receptors.

G protein-coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering of native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized mGluRs (LimGluRs). The light-agonized LimGluR2, on which we focused, was fast, bistable and supported multiple rounds of on/off switching. Light gated two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. We found that the light-antagonized tool LimGluR2-block was able to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalized the optical control to two additional family members: mGluR3 and mGluR6. This system worked in rodent brain slices and in zebrafish in vivo, where we found that mGluR2 modulated the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.

Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors.

Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately 'blind' neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3ß4 and α4ß2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

Robust coding of ego-motion in descending neurons of the fly.

In many species, motion-sensitive neurons responding to optic flow at higher processing stages are well characterized; however, less is known how this representation of ego-motion is further transformed into an appropriate motor response. Here, we analyzed in the blowfly Calliphora vicina the visuomotor transformation from motion-sensitive neurons in the lobula plate [V2 and vertical system (VS) cells] onto premotor descending neurons [descending neurons of the ocellar and vertical system (DNOVS) cells] feeding into the motor circuit of the fly thoracic ganglion. We found that each of these cells is tuned to rotation of the fly around a particular body axis. Comparing the responses of presynaptic and postsynaptic cells revealed that DNOVS cells have approximately the same tuning widths as V2 and VS cells. However, DNOVS signals cells are less corrupted by fluctuations arising from the spatial structure of the visual input than their presynaptic elements. This leads to a more robust representation of ego-motion at the level of descending neurons. Thus, when moving from lobula plate cells to descending neurons, the selectivity for a particular optic flow remains unaltered, but the robustness of the representation increases.

Switching the mechanics of dsDNA by Cu salicylic aldehyde complexation.

DNA is increasingly employed as a programmable building block for nanoscale structures. Self-assembly via specific DNA base-pair recognition allows an unparalleled variety of structures to be formed. Subsequent stabilization of such structures may be desirable and can be accomplished by metal coordination bonds to substituted bases. We investigated the switching of the mechanics of dsDNA carrying salicylic aldehyde nucleosides upon copper complexation. We found the rupture force to increase by up to a factor of two. Furthermore we discovered that the strongly localized coordinative bond dominates the mechanics of this biomolecular hybrid for high loading rates, whereas at lower rates the broad binding potential of the DNA dominates the stability.