Optical mapping and optogenetics in cardiac electrophysiology research and therapy: a state-of-the-art review.

State-of-the-art innovations in optical cardiac electrophysiology are significantly enhancing cardiac research. A potential leap into patient care is now on the horizon. Optical mapping, using fluorescent probes and high-speed cameras, offers detailed insights into cardiac activity and arrhythmias by analysing electrical signals, calcium dynamics, and metabolism. Optogenetics utilizes light-sensitive ion channels and pumps to realize contactless, cell-selective cardiac actuation for modelling arrhythmia, restoring sinus rhythm, and probing complex cell-cell interactions. The merging of optogenetics and optical mapping techniques for 'all-optical' electrophysiology marks a significant step forward. This combination allows for the contactless actuation and sensing of cardiac electrophysiology, offering unprecedented spatial-temporal resolution and control. Recent studies have performed all-optical imaging ex vivo and achieved reliable optogenetic pacing in vivo, narrowing the gap for clinical use. Progress in optical electrophysiology continues at pace. Advances in motion tracking methods are removing the necessity of motion uncoupling, a key limitation of optical mapping. Innovations in optoelectronics, including miniaturized, biocompatible illumination and circuitry, are enabling the creation of implantable cardiac pacemakers and defibrillators with optoelectrical closed-loop systems. Computational modelling and machine learning are emerging as pivotal tools in enhancing optical techniques, offering new avenues for analysing complex data and optimizing therapeutic strategies. However, key challenges remain including opsin delivery, real-time data processing, longevity, and chronic effects of optoelectronic devices. This review provides a comprehensive overview of recent advances in optical mapping and optogenetics and outlines the promising future of optics in reshaping cardiac electrophysiology and therapeutic strategies.

Recent advances in cellular optogenetics for photomedicine.

Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.

Dissection of brain-wide resting-state and functional somatosensory circuits by fMRI with optogenetic silencing.

To further advance functional MRI (fMRI)-based brain science, it is critical to dissect fMRI activity at the circuit level. To achieve this goal, we combined brain-wide fMRI with neuronal silencing in well-defined regions. Since focal inactivation suppresses excitatory output to downstream pathways, intact input and suppressed output circuits can be separated. Highly specific cerebral blood volume-weighted fMRI was performed with optogenetic stimulation of local GABAergic neurons in mouse somatosensory regions. Brain-wide spontaneous somatosensory networks were found mostly in ipsilateral cortical and subcortical areas, which differed from the bilateral homotopic connections commonly observed in resting-state fMRI data. The evoked fMRI responses to somatosensory stimulation in regions of the somatosensory network were successfully dissected, allowing the relative contributions of spinothalamic (ST), thalamocortical (TC), corticothalamic (CT), corticocortical (CC) inputs, and local intracortical circuits to be determined. The ventral posterior thalamic nucleus receives ST inputs, while the posterior medial thalamic nucleus receives CT inputs from the primary somatosensory cortex (S1) with TC inputs. The secondary somatosensory cortex (S2) receives mostly direct CC inputs from S1 and a few TC inputs from the ventral posterolateral nucleus. The TC and CC input layers in cortical regions were identified by laminar-specific fMRI responses with a full width at half maximum of <150 µm. Long-range synaptic inputs in cortical areas were amplified approximately twofold by local intracortical circuits, which is consistent with electrophysiological recordings. Overall, whole-brain fMRI with optogenetic inactivation revealed brain-wide, population-based, long-range circuits, which could complement data typically collected in conventional microscopic functional circuit studies.

Combining Optogenetic Stimulation and Motor Training Improves Functional Recovery and Perilesional Cortical Activity.

Background. An ischemic stroke is followed by the remapping of motor representation and extensive changes in cortical excitability involving both hemispheres. Although stimulation of the ipsilesional motor cortex, especially when paired with motor training, facilitates plasticity and functional restoration, the remapping of motor representation of the single and combined treatments is largely unexplored. Objective. We investigated if spatio-temporal features of motor-related cortical activity and the new motor representations are related to the rehabilitative treatment or if they can be specifically associated to functional recovery. Methods. We designed a novel rehabilitative treatment that combines neuro-plasticizing intervention with motor training. In detail, optogenetic stimulation of peri-infarct excitatory neurons expressing Channelrhodopsin 2 was associated with daily motor training on a robotic device. The effectiveness of the combined therapy was compared with spontaneous recovery and with the single treatments (ie optogenetic stimulation or motor training). Results. We found that the extension and localization of the new motor representations are specific to the treatment, where most treatments promote segregation of the motor representation to the peri-infarct region. Interestingly, only the combined therapy promotes both the recovery of forelimb functionality and the rescue of spatio-temporal features of motor-related activity. Functional recovery results from a new excitatory/inhibitory balance between hemispheres as revealed by the augmented motor response flanked by the increased expression of parvalbumin positive neurons in the peri-infarct area. Conclusions. Our findings highlight that functional recovery and restoration of motor-related neuronal activity are not necessarily coupled during post-stroke recovery. Indeed the reestablishment of cortical activation features of calcium transient is distinctive of the most effective therapeutic approach, the combined therapy.

Enabling comprehensive optogenetic studies of mouse hearts by simultaneous opto-electrical panoramic mapping and stimulation.

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.

Microbial Rhodopsins as Multi-functional Photoreactive Membrane Proteins for Optogenetics.

In life science research, methods to control biological activities with stimuli such as light, heat, pressure and chemicals have been widely utilized to understand their molecular mechanisms. The knowledge obtained by those methods has built a basis for the development of medicinal products. Among those various stimuli, light has the advantage of a high spatiotemporal resolution that allows for the precise control of biological activities. Photoactive membrane protein rhodopsins from microorganisms (called microbial rhodopsins) absorb visible light and that light absorption triggers the trans-cis photoisomerization of the chromophore retinal, leading to various functions such as ion pumps, ion channels, transcriptional regulators and enzymes. In addition to their biological significance, microbial rhodopsins are widely utilized as fundamental molecular tools for optogenetics, a method to control biological activities by light. In this review, we briefly introduce the molecular basis of representative rhodopsin molecules and their applications for optogenetics. Based on those examples, we discuss the high potential of rhodopsin-based optogenetics tools for basic and clinical research in pharmaceutical sciences.

Protocol for targeting the magnocellular neuroendocrine cell ensemble via retrograde tracing from the posterior pituitary.

The hypothalamic magnocellular neuroendocrine cells (MNCs) project to the posterior pituitary (PPi), regulating reproduction and fluid homeostasis. It has been challenging to selectively label and manipulate MNCs, as they are intermingled with parvocellular neuroendocrine cells projecting to the median eminence. Here, we provide a step-by-step protocol for specifically targeting the MNCs by infusing retrograde viral tracers into the PPi. When combined with optogenetics, chemogenetics, and transgenic animals, this approach allows cell-type-specific manipulation of MNCs in multiple sites for functional dissection. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021) and Tang et al. (2020).

Stimulating GABAergic Neurons in the Nucleus Accumbens Core Alters the Trigeminal Neuropathic Pain Responses in a Rat Model of Infraorbital Nerve Injury.

The nucleus accumbens core (NAcc) is an important component of brain reward circuitry, but studies have revealed its involvement in pain circuitry also. However, its effect on trigeminal neuralgia (TN) and the mechanism underlying it are yet to be fully understood. Therefore, this study aimed to examine the outcomes of optogenetic stimulation of NAcc GABAergic neurons in an animal model of TN. Animals were allocated into TN, sham, and control groups. TN was generated by infraorbital nerve constriction and the optogenetic virus was injected into the NAcc. In vivo extracellular recordings were acquired from the ventral posteromedial nucleus of the thalamus. Alterations of behavioral responses during stimulation "ON" and "OFF" conditions were evaluated. In vivo microdialysis was performed in the NAcc of TN and sham animals. During optogenetic stimulation, electrophysiological recordings revealed a reduction of both tonic and burst firing activity in TN animals, and significantly improved behavioral responses were observed as well. Microdialysis coupled with liquid chromatography/tandem mass spectrometry analysis revealed significant alterations in extracellular concentration levels of GABA, glutamate, acetylcholine, dopamine, and citrulline in NAcc upon optic stimulation. In fine, our results suggested that NAcc stimulation could modulate the transmission of trigeminal pain signals in the TN animal model.

Non-canonical role for Lpar1-EGFP subplate neurons in early postnatal mouse somatosensory cortex.

Subplate neurons (SPNs) are thought to play a role in nascent sensory processing in neocortex. To better understand how heterogeneity within this population relates to emergent function, we investigated the synaptic connectivity of Lpar1-EGFP SPNs through the first postnatal week in whisker somatosensory cortex (S1BF). These SPNs comprise of two morphological subtypes: fusiform SPNs with local axons and pyramidal SPNs with axons that extend through the marginal zone. The former receive translaminar synaptic input up until the emergence of the whisker barrels, a timepoint coincident with significant cell death. In contrast, pyramidal SPNs receive local input from the subplate at early ages but then - during the later time window - acquire input from overlying cortex. Combined electrical and optogenetic activation of thalamic afferents identified that Lpar1-EGFP SPNs receive sparse thalamic innervation. These data reveal components of the postnatal network that interpret sparse thalamic input to direct the emergent columnar structure of S1BF.

Conveyance of cortical pacing for parkinsonian tremor-like hyperkinetic behavior by subthalamic dysrhythmia.

Parkinson's disease is characterized by both hypokinetic and hyperkinetic symptoms. While increased subthalamic burst discharges have a direct causal relationship with the hypokinetic manifestations (e.g., rigidity and bradykinesia), the origin of the hyperkinetic symptoms (e.g., resting tremor and propulsive gait) has remained obscure. Neuronal burst discharges are presumed to be autonomous or less responsive to synaptic input, thereby interrupting the information flow. We, however, demonstrate that subthalamic burst discharges are dependent on cortical glutamatergic synaptic input, which is enhanced by A-type K+ channel inhibition. Excessive top-down-triggered subthalamic burst discharges then drive highly correlative activities bottom-up in the motor cortices and skeletal muscles. This leads to hyperkinetic behaviors such as tremors, which are effectively ameliorated by inhibition of cortico-subthalamic AMPAergic synaptic transmission. We conclude that subthalamic burst discharges play an imperative role in cortico-subcortical information relay, and they critically contribute to the pathogenesis of both hypokinetic and hyperkinetic parkinsonian symptoms.

An amygdala-to-hypothalamus circuit for social reward.

Social interactions and relationships are often rewarding, but the neural mechanisms through which social interaction drives positive experience remain poorly understood. In this study, we developed an automated operant conditioning system to measure social reward in mice and found that adult mice of both sexes display robust reinforcement of social interaction. Through cell-type-specific manipulations, we identified a crucial role for GABAergic neurons in the medial amygdala (MeA) in promoting the positive reinforcement of social interaction. Moreover, MeA GABAergic neurons mediate social reinforcement behavior through their projections to the medial preoptic area (MPOA) and promote dopamine release in the nucleus accumbens. Finally, activation of this MeA-to-MPOA circuit can robustly overcome avoidance behavior. Together, these findings establish the MeA as a key node for regulating social reward in both sexes, providing new insights into the regulation of social reward beyond the classic mesolimbic reward system.

Shining light on cardiac electrophysiology: From detection to intervention, from basic research to translational applications.

Cardiac arrhythmias are an important group of cardiovascular diseases, which can occur alone or in association with other cardiovascular diseases. The development of cardiac arrhythmias cannot be separated from changes in cardiac electrophysiology, and the investigation and clarification of cardiac electrophysiological changes are beneficial for the treatment of cardiac arrhythmias. However, electrical energy-based pacemakers and defibrillators, which are widely used to treat arrhythmias, still have certain disadvantages. Thereby, optics promises to be used for optical manipulation and its use in biomedicine is increasing. Since visible light is readily absorbed and scattered in living tissues and tissue penetration is shallow, optical modulation for cells and tissues requires conversion media that convert light energy into bioelectrical activity. In this regard, fluorescent dyes, light-sensitive ion channels, and optical nanomaterials can assume this role, the corresponding optical mapping technology, optogenetics technology, and optical systems based on luminescent nanomaterials have been introduced into the research in cardiovascular field and are expected to be new tools for the study and treatment of cardiac arrhythmias. In addition, infrared and near-infrared light has strong tissue penetration, which is one of the excellent options of external trigger for achieving optical modulation, and is also widely used in the study of optical modulation of biological activities. Here, the advantages of optical applications are summarized, the research progresses and emerging applications of optical-based technologies as detection and intervention tools for cardiac electrophysiological are highlighted. Moreover, the prospects for future applications of optics in clinical diagnosis and treatment are discussed.

Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology.

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.

The Effect of Optogenetic Inhibition of the Anterior Cingulate Cortex in Neuropathic Pain Following Sciatic Nerve Injury.

Cortical disinhibition is the underlying pathological alteration contributing to neuropathic pain associated with peripheral nerve injury. Nerve injury resulting in disinhibition of the anterior cingulate cortex has been reported. However, the effect of optogenetic inhibition of the anterior cingulate cortex (ACC) on the sensory component of nerve injury-induced neuropathic pain has not been well studied. To investigate the feasibility of optogenetic ACC modulation, we injected an optogenetic virus or a null virus into the ACC of a nerve injury-induced neuropathic pain model. The unilateral ACC was modulated, and the optogenetic effect was measured by mechanical and thermal sensitivity tests. The assessment was performed in "pre-light off," "stimulation-yellow light on," and "post-light off" states. Optogenetic inhibition of the ACC in injury models revealed improved mechanical and thermal latencies with profound pain-relieving effects against nerve injury-induced neuropathic pain. The sensory thalamic discharge in electrophysiological in vivo recordings was also altered during laser stimulation. This finding indicates that hyperactivity of the ACC in nerve injury increases output to the spinothalamic tract through direct or indirect pathways. The direct photoinhibition of ACC neurons could play a vital role in restoring equilibrium and provide novel insight into techniques that can assuage peripheral nerve injury-induced neuropathic pain.

Imaging and optogenetic modulation of vascular mural cells in the live brain.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.

[Vision restoration: science fiction or reality?] / Restauration de la vision: Science-fiction ou réalité ?

Visual prostheses aim at restoring useful vision to patients who have become blind. This useful vision should enable them to regain autonomy in society for navigation, face recognition or reading. Two retinal prostheses have already obtained market authorization for patients affected by retinal dystrophies while a new device is in clinical trials for patients affected by age-related macular degeneration. Various prostheses, in particular cortical prostheses, are currently in clinical trials for optic neuropathies (glaucoma). Optogenetic therapy, an alternative strategy, has now reached the stage of clinical trials at the retinal level while moving forward at the cortical level. Other innovating strategies have obtained proofs of concepts in rodents but require a further validation in large animals prior to their evaluation on patients. Restoring vision should therefore become a reality for many patients even if this vision will not be as extensive and perfect as natural vision.

TITLE: Restauration de la vision: Science-fiction ou réalité ? ABSTRACT: Les prothèses visuelles ont pour objet de redonner une vision utile aux patients devenus aveugles. Cette vision utile doit leur permettre de retrouver une autonomie dans la société pour leurs déplacements, la reconnaissance des visages ou la lecture. Plusieurs prothèses rétiniennes ont déjà obtenu l'autorisation de mise sur le marché pour les dystrophies rétiniennes alors qu'un nouveau dispositif est en essai clinique pour la dégénérescence maculaire liée à l'âge. D'autres prothèses, notamment corticales, sont en essai clinique pour les neuropathies optiques (glaucome). Des stratégies alternatives, comme la thérapie optogénétique, ont également atteint le stade des essais cliniques. D'autres ont été évaluées sur les rongeurs, attendant leur validation sur le gros animal. Revoir devrait donc prochainement devenir une réalité pour de nombreux patients, même si cette vision ne sera ni aussi étendue, ni aussi parfaite que la vision naturelle.

Pain modulation effect on motor cortex after optogenetic stimulation in shPKCγ knockdown dorsal root ganglion-compressed Sprague-Dawley rat model.

Neuropathic pain can be generated by chronic compression of dorsal root ganglion (CCD). Stimulation of primary motor cortex can disrupt the nociceptive sensory signal at dorsal root ganglion level and reduce pain behaviors. But the mechanism behind it is still implicit. Protein kinase C gamma is known as an essential enzyme for the development of neuropathic pain, and specific inhibitor of protein kinase C gamma can disrupt the sensory signal and reduce pain behaviors. Optogenetic stimulation has been emerged as a new and promising conducive method for refractory neuropathic pain. The aim of this study was to provide evidence whether optical stimulation of primary motor cortex can modulate chronic neuropathic pain in CCD rat model. Animals were randomly divided into CCD group, sham group, and control group. Dorsal root ganglion-compressed neuropathic pain model was established in animals, and knocking down of protein kinase C gamma was also accomplished. Pain behavioral scores were significantly improved in the short hairpin Protein Kinase C gamma knockdown CCD animals during optic stimulation. Ventral posterolateral thalamic firing inhibition was also observed during light stimulation on motor cortex in CCD animal. We assessed alteration of pain behaviors in pre-light off, stimulation-light on, and post-light off state. In vivo extracellular recording of the ventral posterolateral thalamus, viral expression in the primary motor cortex, and protein kinase C gamma expression in dorsal root ganglion were investigated. So, optical cortico-thalamic inhibition by motor cortex stimulation can improve neuropathic pain behaviors in CCD animal, and knocking down of protein kinase C gamma plays a conducive role in the process. This study provides feasibility for in vivo optogenetic stimulation on primary motor cortex of dorsal root ganglion-initiated neuropathic pain.

Ultraflexible organic light-emitting diodes for optogenetic nerve stimulation.

Organic electronic devices implemented on flexible thin films are attracting increased attention for biomedical applications because they possess extraordinary conformity to curved surfaces. A neuronal device equipped with an organic light-emitting diode (OLED), used in combination with animals that are genetically engineered to include a light-gated ion channel, would enable cell type-specific stimulation to neurons as well as conformal contact to brain tissue and peripheral soft tissue. This potential application of the OLEDs requires strong luminescence, well over the neuronal excitation threshold in addition to flexibility. Compatibility with neuroimaging techniques such as MRI provides a method to investigate the evoked activities in the whole brain. Here, we developed an ultrathin, flexible, MRI-compatible OLED device and demonstrated the activation of channelrhodopsin-2-expressing neurons in animals. Optical stimulation from the OLED attached to nerve fibers induced contractions in the innervated muscles. Mechanical damage to the tissues was significantly reduced because of the flexibility. Owing to the MRI compatibility, neuronal activities induced by direct optical stimulation of the brain were visualized using MRI. The OLED provides an optical interface for modulating the activity of soft neuronal tissues.

Mapping Functional Connectivity from the Dorsal Cortex to the Thalamus.

Neural activity in the corticothalamic network is crucial for sensation, memory, decision, and action. Nevertheless, a systematic characterization of corticothalamic functional connectivity has not been achieved. Here, we developed a high throughput method to systematically map functional connections from the dorsal cortex to the thalamus in awake mice by combing optogenetic inactivation with multi-channel recording. Cortical inactivation resulted in a rapid reduction of thalamic activity, revealing topographically organized corticothalamic excitatory inputs. Cluster analysis showed that groups of neurons within individual thalamic nuclei exhibited distinct dynamics. The effects of inactivation evolved with time and were modulated by behavioral states. Furthermore, we found that a subset of thalamic neurons received convergent inputs from widespread cortical regions. Our results present a framework for collecting, analyzing, and presenting large electrophysiological datasets with region-specific optogenetic perturbations and serve as a foundation for further investigation of information processing in the corticothalamic pathway.

Towards the clinical translation of optogenetic skeletal muscle stimulation.

Paralysis is a frequent phenomenon in many diseases, and to date, only functional electrical stimulation (FES) mediated via the innervating nerve can be employed to restore skeletal muscle function in patients. Despite recent progress, FES has several technical limitations and significant side effects. Optogenetic stimulation has been proposed as an alternative, as it may circumvent some of the disadvantages of FES enabling cell type-specific, spatially and temporally precise stimulation of cells expressing light-gated ion channels, commonly Channelrhodopsin2. Two distinct approaches for the restoration of skeletal muscle function with optogenetics have been demonstrated: indirect optogenetic stimulation through the innervating nerve similar to FES and direct optogenetic stimulation of the skeletal muscle. Although both approaches show great promise, both have their limitations and there are several general hurdles that need to be overcome for their translation into clinics. These include successful gene transfer, sustained optogenetic protein expression, and the creation of optically active implantable devices. Herein, a comprehensive summary of the underlying mechanisms of electrical and optogenetic approaches is provided. With this knowledge in mind, we substantiate a detailed discussion of the advantages and limitations of each method. Furthermore, the obstacles in the way of clinical translation of optogenetic stimulation are discussed, and suggestions on how they could be overcome are provided. Finally, four specific examples of pathologies demanding novel therapeutic measures are discussed with a focus on the likelihood of direct versus indirect optogenetic stimulation.