Optogenetics combines optics and genetic engineering to control specific gene expression and biological functions and has the advantages of precise spatiotemporal control, noninvasiveness, and high efficiency. Genetically modified photosensory sensors are engineered into proteins to modulate conformational changes with light stimulation. Therefore, optogenetic techniques can provide new insights into oral biological processes at different levels, ranging from the subcellular and cellular levels to neural circuits and behavioral models. Here, we introduce the origins of optogenetics and highlight the recent progress of optogenetic approaches in oral and craniofacial research, focusing on the ability to apply optogenetics to the study of basic scientific neural mechanisms and to establish different oral behavioral test models in vivo (orofacial movement, licking, eating, and drinking), such as channelrhodopsin (ChR), archaerhodopsin (Arch), and halorhodopsin from Natronomonas pharaonis (NpHR). We also review the synergic and antagonistic effects of optogenetics in preclinical studies of trigeminal neuralgia and maxillofacial cellulitis. In addition, optogenetic tools have been used to control the neurogenic differentiation of dental pulp stem cells in translational studies. Although the scope of optogenetic tools is increasing, there are limited large animal experiments and clinical studies in dental research. Potential future directions include exploring therapeutic strategies for addressing loss of taste in patients with coronavirus disease 2019 (COVID-19), studying oral bacterial biofilms, enhancing craniomaxillofacial and periodontal tissue regeneration, and elucidating the possible pathogenesis of dry sockets, xerostomia, and burning mouth syndrome.
光遗传学通过光学和基因工程相结合对特定的基因表达和生物功能进行控制，具有精确的时空控制、无创和高效等优点。蛋白质被植入经基因改造的光感传感器后，在光刺激下调节构象的变化。因此，光遗传学技术可在从亚细胞和细胞水平到神经回路和行为模型等不同层面上为口腔生物学研究提供新的见解。本综述介绍了光遗传学的起源和光遗传学方法在口腔颌面研究中的最新进展，着重描述了通道视紫红质（ChR）、古紫质（Arch）和氯视紫红质（NpHR）等光遗传学工具在神经科学基础机制研究中的应用，以及在体内建立不同的口腔行为测试模型（口面部运动、舔舐、进食和饮水），同时回顾了光遗传学在三叉神经痛和颌面蜂窝织炎的临床前研究中的协同和拮抗作用。此外，在转化研究中，光遗传学工具被用于控制牙髓干细胞的神经源性分化。虽然光遗传学工具的应用范围在不断扩大，但其在口腔研究领域的大型动物实验和临床研究中的应用还很有限。光遗传学潜在的应用方向包括探索2019冠状病毒病（COVID-19）患者味觉丧失的治疗策略、研究口腔细菌生物膜、增强颅颌面和牙周组织再生，以及阐明干槽症、口干症和灼口综合征的可能发病机制。.

To understand the function of cells such as neurons within an organism, it can be instrumental to inhibit cellular function, or to remove the cell (type) from the organism, and thus to observe the consequences on organismic and/or circuit function and animal behavior. A range of approaches and tools were developed and used over the past few decades that act either constitutively or acutely and reversibly, in systemic or local fashion. These approaches make use of either drugs or genetically encoded tools. Also, there are acutely acting inhibitory tools that require an exogenous trigger like light. Here, we give an overview of such methods developed and used in the nematode Caenorhabditis elegans.

All-optical methods that provide deeper understanding of neural activity are currently being developed. Optogenetics is a biological technique useful to control neuronal activity or life phenomena using light. Microbial rhodopsins are light-activated membrane proteins used as optogenetic tools. Microbial rhodopsins such as channelrhodopsin2 (ChR2) consist of seven-pass transmembrane proteins with a covalently bound retinal. Light absorption is followed by photoisomerization of the all-trans retinal to a 13-cis configuration and subsequent conformational changes in the molecule, with consequent permeability of the channel structure to ions. Recent studies have reported the discovery of microbial rhodopsins with novel functions. Microbial rhodopsin diversity has also increased. We describe the characteristics of microbial rhodopsins used as optogenetic tools and the latest research in this domain.

Transient nigrostriatal dopaminergic signalling is well known for its role in reinforcement learning and increasingly so for its role in the initiation of voluntary movement. However, how transient bursts of dopamine modulate voluntary movement remains unclear, likely due to the heterogeneity of the nigrostriatal system, the focus of optogenetic studies on locomotion at sub-sec time intervals, and the overlapping roles of phasic dopamine in behaviour and novelty signalling. In this study we investigated how phasic activity in the lateral substantia nigra pars compacta (lateral SNc) over time affects voluntary behaviours during exploration. Using a transgenic mouse model of both sexes expressing channelrhodopsin (ChR2) in dopamine transporter-expressing cells, we stimulated the lateral SNc while mice explored an open field over two consecutive days. We found that phasic activation of the lateral SNc induced an increase in exploratory behaviours including horizontal movement activity, locomotion initiation, and rearing specifically on the first open field exposure, but not on the second day. In addition, stimulated animals did not habituate to the same extent as their ChR2-negative counterparts, as indicated by a lack of decrease in baseline activity. These findings suggest that rather than prompting voluntary movement in general, phasic nigrostriatal dopamine prompts context-appropriate behaviours. In addition, dopamine signalling that modulates movement acts over longer timescales than the transient signal, affecting behaviour even after the signal has ended.

Channelrhodopsin (ChR) and heliorhodopsin (HeR) are microbial rhodopsins with similar structures but different circular dichroism (CD) spectra: ChR shows biphasic negative and positive bands, whereas HeR shows a single positive band. We explored the physicochemical factors underlying these differences through computational methods. Using the exciton model based on first-principles computations, we obtained the CD spectra of ChR and HeR. The obtained spectra indicate that the protein dimer structures and the quantum mechanical treatment of the retinal chromophore and its interacting amino acids are crucial for accurately reproducing the experimental spectra. Further calculations revealed that the sign of the excitonic coupling was opposite between the ChR and HeR dimers, which was attributed to the contrasting second term of the orientation factor between the two retinal chromophores. These findings demonstrate that slight variations in the intermolecular orientation of the two chromophores can result in significant differences in the CD spectral shape.

The light-gated anion channelrhodopsin GtACR1 is an important optogenetic tool for neuronal silencing. Its photochemistry, including its photointermediates, is poorly understood. The current mechanistic view presumes BR-like kinetics and assigns the open channel to a blue-absorbing L intermediate. Based on time-resolved absorption and electrophysiological data, we recently proposed a red-absorbing spectral form for the open channel state. Here, we report the results of a comprehensive kinetic analysis of the spectroscopic data combined with channel current information. The time evolutions of the spectral forms derived from the spectroscopic data are inconsistent with the single chain mechanism and are analyzed within the concept of parallel photocycles. The spectral forms partitioned into conductive and nonconductive parallel cycles are assigned to intermediate states. Rejecting reversible connections between conductive and nonconductive channel states leads to kinetic schemes with two independent conductive states corresponding to the fast- and slow-decaying current components. The conductive cycle is discussed in terms of a single cycle and two parallel cycles. The reaction mechanisms and reaction rates for the wild-type protein, the A75E, and the low-conductance D234N and S97E protein variants are derived. The parallel cycles of channelrhodopsin kinetics, its relation to BR photocycle, and the role of the M intermediate in channel closure are discussed.

Mitral/tufted cells (M/TCs) form complex local circuits with interneurons in the olfactory bulb and are powerfully inhibited by these interneurons. The horizontal limb of the diagonal band of Broca (HDB), the only GABAergic/inhibitory source of centrifugal circuit with the olfactory bulb, is known to target olfactory bulb interneurons, and we have shown targeting also to olfactory bulb glutamatergic neurons in vitro. However, the net efficacy of these circuits under different patterns of activation in vivo and the relative balance between the various targeted intact local and centrifugal circuits was the focus of this study. Here channelrhodopsin-2 (ChR2) was expressed in HDB GABAergic neurons to investigate the short-term plasticity of HDB-activated disinhibitory rebound excitation of M/TCs. Optical activation of HDB interneurons increased spontaneous M/TC firing without odor presentation and increased odor-evoked M/TC firing. HDB activation induced disinhibitory rebound excitation (burst or cluster of spiking) in all classes of M/TCs. This excitation was frequency dependent, with short-term facilitation only at higher HDB stimulation frequency (5 Hz and above). However, frequency-dependent HDB regulation was more potent in the deeper layer M/TCs compared with more superficial layer M/TCs. In all neural circuits the balance between inhibition and excitation in local and centrifugal circuits plays a critical functional role, and this patterned input-dependent regulation of inhibitory centrifugal inputs to the olfactory bulb may help maintain the precise balance across the populations of output neurons in different environmental odors, putatively to sharpen the enhancement of tuning specificity of individual or classes of M/TCs to odors.NEW & NOTEWORTHY Neuronal local circuits in the olfactory bulb are modulated by centrifugal long circuits. In vivo study here shows that inhibitory horizontal limb of the diagonal band of Broca (HDB) modulates all five types of mitral/tufted cells (M/TCs), by direct inhibitory circuits HDB → M/TCs and indirect disinhibitory long circuits HDB → interneurons → M/TCs. The HDB net effect exerts excitation in all types of M/TCs but more powerful in deeper layer output neurons as HDB activation frequency increases, which may sharpen the tuning specificity of classes of M/TCs to odors during sensory processing.

Hearing loss is a prevalent sensory impairment significantly affecting communication and quality of life. Traditional approaches for hearing restoration, such as cochlear implants, have limitations in frequency resolution and spatial selectivity. Optogenetics, an emerging field utilizing light-sensitive proteins, offers a promising avenue for addressing these limitations and revolutionizing hearing rehabilitation. This review explores the methods of introducing Channelrhodopsin- 2 (ChR2), a key light-sensitive protein, into cochlear cells to enable optogenetic stimulation. Viral- mediated gene delivery is a widely employed technique in optogenetics. Selecting a suitable viral vector, such as adeno-associated viruses (AAV), is crucial in efficient gene delivery to cochlear cells. The ChR2 gene is inserted into the viral vector through molecular cloning techniques, and the resulting viral vector is introduced into cochlear cells via direct injection or round window membrane delivery. This allows for the expression of ChR2 and subsequent light sensitivity in targeted cells. Alternatively, direct cell transfection offers a non-viral approach for ChR2 delivery. The ChR2 gene is cloned into a plasmid vector, which is then combined with transfection agents like liposomes or nanoparticles. This mixture is applied to cochlear cells, facilitating the entry of the plasmid DNA into the target cells and enabling ChR2 expression. Optogenetic stimulation using ChR2 allows for precise and selective activation of specific neurons in response to light, potentially overcoming the limitations of current auditory prostheses. Moreover, optogenetics has broader implications in understanding the neural circuits involved in auditory processing and behavior. The combination of optogenetics and gene delivery techniques provides a promising avenue for improving hearing restoration strategies, offering the potential for enhanced frequency resolution, spatial selectivity, and improved auditory perception.

We recently reported that the central nucleus of the inferior colliculus (the auditory midbrain) is innervated by glutamatergic pyramidal cells originating not only in auditory cortex (AC), but also in multiple \'non-auditory\' regions of the cerebral cortex. Here, in anaesthetised rats, we used optogenetics and electrical stimulation, combined with recording in the inferior colliculus to determine the functional influence of these descending connections. Specifically, we determined the extent of monosynaptic excitation and the influence of these descending connections on spontaneous activity in the inferior colliculus. A retrograde virus encoding both green fluorescent protein (GFP) and channelrhodopsin (ChR2) injected into the central nucleus of the inferior colliculus (ICc) resulted in GFP expression in discrete groups of cells in multiple areas of the cerebral cortex. Light stimulation of AC and primary motor cortex (M1) caused local activation of cortical neurones and increased the firing rate of neurones in ICc indicating a direct excitatory input from AC and M1 to ICc with a restricted distribution. In naïve animals, electrical stimulation at multiple different sites within M1, secondary motor, somatosensory, and prefrontal cortices increased firing rate in ICc. However, it was notable that stimulation at some adjacent sites failed to influence firing at the recording site in ICc. Responses in ICc comprised singular spikes of constant shape and size which occurred with a short, and fixed latency (∼ 5 ms) consistent with monosynaptic excitation of individual ICc units. Increasing the stimulus current decreased the latency of these spikes, suggesting more rapid depolarization of cortical neurones, and increased the number of (usually adjacent) channels on which a monosynaptic spike was seen, suggesting recruitment of increasing numbers of cortical neurons. Electrical stimulation of cortical regions also evoked longer latency, longer duration increases in firing activity, comprising multiple units with spikes occurring with significant temporal jitter, consistent with polysynaptic excitation. Increasing the stimulus current increased the number of spikes in these polysynaptic responses and increased the number of channels on which the responses were observed, although the magnitude of the responses always diminished away from the most activated channels. Together our findings indicate descending connections from motor, somatosensory and executive cortical regions directly activate small numbers of ICc neurones and that this in turn leads to extensive polysynaptic activation of local circuits within the ICc.

Soft bioelectronic devices exhibit motion-adaptive properties for neural interfaces to investigate complex neural circuits. Here, we develop a fabrication approach through the control of metamorphic polymers\' amorphous-crystalline transition to miniaturize and integrate multiple components into hydrogel bioelectronics. We attain an about 80% diameter reduction in chemically cross-linked polyvinyl alcohol hydrogel fibers in a fully hydrated state. This strategy allows regulation of hydrogel properties, including refractive index (1.37-1.40 at 480 nm), light transmission (>96%), stretchability (139-169%), bending stiffness (4.6 ± 1.4 N/m), and elastic modulus (2.8-9.3 MPa). To exploit the applications, we apply step-index hydrogel optical probes in the mouse ventral tegmental area, coupled with fiber photometry recordings and social behavioral assays. Additionally, we fabricate carbon nanotubes-PVA hydrogel microelectrodes by incorporating conductive nanomaterials in hydrogel for spontaneous neural activities recording. We enable simultaneous optogenetic stimulation and electrophysiological recordings of light-triggered neural activities in Channelrhodopsin-2 transgenic mice.