Broadband nonlinear optical response and sub-picosecond carrier dynamics in graphene-SnSe2 van der Waals heterostructures.

The van der Waals (vdWs) heterostructures, with vertical layer stacking structure of various two-dimensional (2D) materials, maintain the reliable photonic characteristics while compensating the shortcomings of the participating individual components. In this work, we combine the less-studied multilayer tin selenide (SnSe2) thin film with one of the traditional 2D materials, graphene, to fabricate the graphene-based vdWs optical switching element (Gr-SnSe2) with superior broadband nonlinear optical response. The transient absorption spectroscopy (TAS) measurement results verify that graphene acts as the recombination channel for the photogenerated carrier in the Gr-SnSe2 sample, and the fast recovery time can be reduced to hundreds of femtoseconds which is beneficial for the optical modulation process. The optical switching properties are characterized by the I-scan measurements, exhibiting a saturable energy intensity of 2.82 mJ·cm-2 (0.425 µJ·cm-2) and a modulation depth of 15.6% (22.5%) at the wavelength of 1030 nm (1980nm). Through integrating Gr-SnSe2 with a cladding waveguide, high-performance picosecond Q-switched operation in the near-infrared (NIR) and mid-infrared (MIR) spectral regions are both achieved. This work experimentally demonstrates the great potential of graphene-based vdWs heterostructures for applications in broadband ultrafast photonics.

Estrogens rapidly shape synaptic and intrinsic properties to regulate the temporal precision of songbird auditory neurons.

Sensory neurons parse millisecond-variant sound streams like birdsong and speech with exquisite precision. The auditory pallial cortex of vocal learners like humans and songbirds contains an unconventional neuromodulatory system: neuronal expression of the estrogen synthesis enzyme aromatase. Local forebrain neuroestrogens fluctuate when songbirds hear a song, and subsequently modulate bursting, gain, and temporal coding properties of auditory neurons. However, the way neuroestrogens shape intrinsic and synaptic properties of sensory neurons remains unknown. Here, using a combination of whole-cell patch clamp electrophysiology and calcium imaging, we investigate estrogenic neuromodulation of auditory neurons in a region resembling mammalian auditory association cortex. We found that estradiol rapidly enhances the temporal precision of neuronal firing via a membrane-bound G-protein coupled receptor and that estradiol rapidly suppresses inhibitory synaptic currents while sparing excitation. Notably, the rapid suppression of intrinsic excitability by estradiol was predicted by membrane input resistance and was observed in both males and females. These findings were corroborated by analysis of in vivo electrophysiology recordings, in which local estrogen synthesis blockade caused acute disruption of the temporal correlation of song-evoked firing patterns. Therefore, on a modulatory timescale, neuroestrogens alter intrinsic cellular properties and inhibitory neurotransmitter release to regulate the temporal precision of higher-order sensory neurons.

P2X7 receptor is required for the ototoxicity caused by aminoglycoside in developing cochlear hair cells.

Aminoglycoside antibiotics (AGAs) are widely used in life-threatening infections, but they accumulate in cochlear hair cells (HCs) and result in hearing loss. Increases in adenosine triphosphate (ATP) concentrations and P2X7 receptor expression were observed after neomycin treatment. Here, we demonstrated that P2X7 receptor, which is a non-selective cation channel that is activated by high ATP concentrations, may participate in the process through which AGAs enter hair cells. Using transgenic knockout mice, we found that P2X7 receptor deficiency protects HCs against neomycin-induced injury in vitro and in vivo. Subsequently, we used fluorescent gentamicin-Fluor 594 to study the uptake of AGAs and found fluorescence labeling in wild-type mice but not in P2rx7-/- mice in vitro. In addition, knocking-out P2rx7 did not significantly alter the HC count and auditory signal transduction, but it did inhibit mitochondria-dependent oxidative stress and apoptosis in the cochlea after neomycin exposure. We thus conclude that the P2X7 receptor may be linked to the entry of AGAs into HCs and is likely to be a therapeutic target for auditory HC protection.

Gene editing in a Myo6 semi-dominant mouse model rescues auditory function.

Myosin VI（MYO6） is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6WT/C442Y mouse model that recapitulated the phenotypes of human patients. The in vivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6C442Y is 4.05% on average in Myo6WT/C442Y mice, which was ∼17-fold greater than editing efficiency of Myo6WT alleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6WT/C442Y mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.

A humanized murine model, demonstrating dominant progressive hearing loss caused by a novel KCNQ4 mutation (p.G228D) from a large Chinese family.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Two-dimensional Ti3C2Tx MXene promotes electrophysiological maturation of neural circuits.

BACKGROUND: The ideal neural interface or scaffold for stem cell therapy shall have good biocompatibility promoting survival, maturation and integration of neural stem cells (NSCs) in targeted brain regions. The unique electrical, hydrophilic and surface-modifiable properties of Ti3C2Tx MXene make it an attractive substrate, but little is known about how it interacts with NSCs during development and maturation. RESULTS: In this study, we cultured NSCs on Ti3C2Tx MXene and examined its effects on morphological and electrophysiological properties of NSC-derived neurons. With a combination of immunostaining and patch-clamp recording, we found that Ti3C2Tx MXene promotes NSCs differentiation and neurite growth, increases voltage-gated current of Ca2+ but not Na+ or K+ in matured neurons, boosts their spiking without changing their passive membrane properties, and enhances synaptic transmission between them. CONCLUSIONS: These results expand our understanding of interaction between Ti3C2Tx MXene and NSCs and provide a critical line of evidence for using Ti3C2Tx MXene in neural interface or scaffold in stem cell therapy.

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

Memristors with diffusive dynamics as synaptic emulators for neuromorphic computing.

The accumulation and extrusion of Ca2+ in the pre- and postsynaptic compartments play a critical role in initiating plastic changes in biological synapses. To emulate this fundamental process in electronic devices, we developed diffusive Ag-in-oxide memristors with a temporal response during and after stimulation similar to that of the synaptic Ca2+ dynamics. In situ high-resolution transmission electron microscopy and nanoparticle dynamics simulations both demonstrate that Ag atoms disperse under electrical bias and regroup spontaneously under zero bias because of interfacial energy minimization, closely resembling synaptic influx and extrusion of Ca2+, respectively. The diffusive memristor and its dynamics enable a direct emulation of both short- and long-term plasticity of biological synapses, representing an advance in hardware implementation of neuromorphic functionalities.

Probing electrical tuning of hair cells with a Zap current method in the intact amphibian papilla of bullfrogs.

Most, if not all, modern vertebrate species have evolved exquisite inner ears to discriminate acoustic signals of different frequencies, through a process called frequency tuning. For non-mammalian species, at least part of frequency tuning has been attributed to intrinsic electrical properties of hair cells, i.e. electrical tuning. Since it was first discovered, the traditional method to assess electrical tuning has been to inject step current into hair cells and examine dampened membrane voltage oscillation. However, this method is not applicable for hair cells that do not oscillate. In this study, we developed a Zap current method that can be unbiasedly applied to all hair cells regardless of their oscillating behavior. Similar to a chirp sound in acoustic stimulation, a Zap current is a sinusoidal current with the frequency increased linearly with time. We first validated this new method with the traditional step current method on hair cells with dampened membrane voltage oscillation, and then applied it to all hair cells in the intact amphibian papilla of bullfrogs. We found that while hair cells with dampened membrane voltage oscillation are sharply tuned, non-oscillating hair cells are broadly tuned. In addition, we found a third type of hair cells, which oscillate continuously and are extremely sharply tuned, with multiple peaks that are reminiscent of harmonics in the mammalian cochlea. In conclusion, the new Zap current method provides an unbiased way to assess electrical tuning, and it reveals an underappreciated heterogeneity of electrical tuning in the bullfrog amphibian papilla.

Danshensu reduces neuronal excitability by enhancing potassium currents in bushy cells in the mouse cochlear nucleus.

Danshensu, also known as salvianic acid A, is a primary active compound extracted from a traditional Chinese herb Danshen (Salvia miltiorrhiza). While its antioxidative and neuroprotective effects are well-documented, the underlying mechanisms are poorly understood. In this study, we sought out to investigate if and how Danshensu modulates neuronal excitability and voltage-gated ionic currents in the central nervous system. We prepared brain slices of the mouse brainstem and performed patch-clamp recording in bushy cells in the anteroventral cochlear nucleus, with or without Danshensu incubation for 1 h. QX-314 was used internally to block Na+ current, while tetraethylammonium and 4-aminopyridine were used to isolate different subtypes of K+ current. We found that Danshensu of 100 µm decreased the input resistance of bushy cells by approximately 60% and shifted the voltage threshold of spiking positively by approximately 7 mV, resulting in significantly reduced excitability. Furthermore, we found this reduced excitability by Danshensu was caused by enhanced voltage-gated K+ currents in these neurons, including both low voltage-activated IK,A, by approximately 100%, and high voltage-activated IK,dr, by approximately 30%. Lastly, we found that the effect of Danshensu on K+ currents was dose-dependent in that no enhancement was found for Danshensu of 50 µm and Danshensu of 200 µm failed to cause significantly more enhancement on K+ currents when compared to that of 100 µm. We found that Danshensu reduced neuronal excitability in the central nervous system by enhancing voltage-gated K+ currents, providing mechanistic support for its neuroprotective effect widely seen in vivo.

Bionic visual-audio photodetectors with in-sensor perception and preprocessing.

Serving as the "eyes" and "ears" of the Internet of Things, optical and acoustic sensors are the fundamental components in hardware systems. Nowadays, mainstream hardware systems, often comprising numerous discrete sensors, conversion modules, and processing units, tend to result in complex architectures that are less efficient compared to human sensory pathways. Here, a visual-audio photodetector inspired by the human perception system is proposed to enable all-in-one visual and acoustic signal detection with computing capability. This device not only captures light but also optically records sound waves, thus achieving "watching" and "listening" within a single unit. The gate-tunable positive, negative, and zero photoresponses lead to highly programmable responsivities. This programmability enables the execution of diverse functions, including visual feature extraction, object classification, and sound wave manipulation. These results showcase the potential of expanding perception approaches in neuromorphic devices, opening up new possibilities to craft intelligent and compact hardware systems.

Schottky infrared detectors with optically tunable barriers beyond the internal photoemission limit.

Internal photoemission is a prominent branch of the photoelectric effect and has emerged as a viable method for detecting photons with energies below the semiconductor bandgap. This breakthrough has played a significant role in accelerating the development of infrared imaging in one chip with state-of-the-art silicon techniques. However, the performance of these Schottky infrared detectors is currently hindered by the limit of internal photoemission; specifically, a low Schottky barrier height is inevitable for the detection of low-energy infrared photons. Herein, a distinct paradigm of Schottky infrared detectors is proposed to overcome the internal photoemission limit by introducing an optically tunable barrier. This device uses an infrared absorbing material-sensitized Schottky diode, assisted by the highly adjustable Fermi level of graphene, which subtly decouples the photon energy from the Schottky barrier height. Correspondingly, a broadband photoresponse spanning from ultraviolet to mid-wave infrared is achieved, with a high specific detectivity of 9.83 × 1010 cm Hz1/2 W-1 at 2,700 nm and an excellent specific detectivity of 7.2 × 109 cm Hz1/2 W-1 at room temperature under blackbody radiation. These results address a key challenge in internal photoemission and hold great promise for the development of the Schottky infrared detector with high sensitivity and room temperature operation.

Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear.

Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.

Hair cell-specific Myo15 promoter-mediated gene therapy rescues hearing in DFNB9 mouse model.

Adeno-associated viral (AAV) vectors are increasingly used as vehicles for gene delivery to treat hearing loss. However, lack of specificity of the transgene expression may lead to overexpression of the transgene in nontarget tissues. In this study, we evaluated the expression efficiency and specificity of transgene delivered by AAV-PHP.eB under the inner ear sensory cell-specific Myo15 promoter. Compared with the ubiquitous CAG promoter, the Myo15 promoter initiates efficient expression of the GFP fluorescence reporter in hair cells, while minimizing non-specific expression in other cell types of the inner ear and CNS. Furthermore, using the Myo15 promoter, we constructed an AAV-mediated therapeutic system with the coding sequence of OTOF gene. After inner ear injection, we observed apparent hearing recovery in Otof-/- mice, highly efficient expression of exogenous otoferlin, and significant improvement in the exocytosis function of inner hair cells. Overall, our results indicate that gene therapy mediated by the hair cell-specific Myo15 promoter has potential clinical application for the treatment of autosomal recessive deafness and yet for other hereditary hearing loss related to dysfunction of hair cells.

Single Ca2+ channels and exocytosis at sensory synapses.

Hair cell synapses in the ear and photoreceptor synapses in the eye are the first synapses in the auditory and visual system. These specialized synapses transmit a large amount of sensory information in a fast and efficient manner. Moreover, both small and large signals with widely variable kinetics must be quickly encoded and reliably transmitted to allow an animal to rapidly monitor and react to its environment. Here we briefly review some aspects of these primary synapses, which are characterized by a synaptic ribbon in their active zones of transmitter release. We propose that these synapses are themselves highly specialized for the task at hand. Photoreceptor and bipolar cell ribbon synapses in the retina appear to have versatile properties that permit both tonic and phasic transmitter release. This allows them to transmit changes of both luminance and contrast within a visual field at different ambient light levels. By contrast, hair cell ribbon synapses are specialized for a highly synchronous form of multivesicular release that may be critical for phase locking to low-frequency sound-evoked signals at both low and high sound intensities. The microarchitecture of a hair cell synapse may be such that the opening of a single Ca(2+) channel evokes the simultaneous exocytosis of multiple synaptic vesicles. Thus, the differing demands of sensory encoding in the eye and ear generate diverse designs and capabilities for their ribbon synapses.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction.

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Probing surface properties of organic molecular layers by scanning tunneling microscopy.

In view of the relevance of organic thin layers in many fields, the fundamentals, growth mechanisms, and dynamics of thin organic layers, in particular thiol-based self-assembled monolayers (SAMs) on Au(111) are systematically elaborated. From both theoretical and practical perspectives, dynamical and structural features of the SAMs are of great intrigue. Scanning tunneling microscopy (STM) is a remarkably powerful technique employed in the characterization of SAMs. Numerous research examples of investigation about the structural and dynamical properties of SAMs using STM, sometimes combined with other techniques, are listed in the review. Advanced options to enhance the time resolution of STM are discussed. Additionally, we elaborate on the extremely diverse dynamics of various SAMs, such as phase transitions and structural changes at the molecular level. In brief, the current review is expected to supply a better understanding and novel insights regarding the dynamical events happening in organic SAMs and how to characterize these processes.

Graphene/Ge Photoconductive Position-Sensitive Detectors Based on the Charge Injection Effect.

Position-sensitive detectors (PSDs) are of great significance to optical communication, automatic alignment, and dislocation detection domains, by precisely obtaining the position information of infrared light spots which are invisible to human eyes. Herein, a kind of PSD based on graphene/germanium (Ge) heterojunction architecture is proposed and demonstrated, which exhibits amplified signals by unitizing the charge injection effect. Driven by the graphene/Ge heterojunction, a large number of photogenerated carriers diffuse from the incident position of the light spot and subsequently inject into graphene, which ultimately generates a photoresponse with high efficiency. The experimental results show that the device can exhibit a fast response speed of 3 µs, a high responsivity of ~40 A/W, and a detection distance of 3000 µm at the 1550 nm band, which hints that the graphene/Ge heterojunction can be used as an efficient platform for near-infrared light spot position sensing.