Else Kröner Fresenius Center for Optogenetic Therapies

Optogenetic control is used to manipulate the activity of specific cell types in vivo for a variety of biological and clinical applications. Here we report ChReef, an improved variant of the channelrhodopsin ChRmine. ChReef offers minimal photocurrent desensitization, a unitary conductance of 80 fS and closing kinetics of 30 ms, which together enable reliable optogenetic control of cells at low light levels with good temporal fidelity and sustained stimulation. We demonstrate efficient and reliable red-light pacing and depolarization block of ChReef-expressing cardiomyocyte clusters. We used adeno-associated-virus-based gene transfer to express ChReef in retinal ganglion cells, where it restores visual function in blind mice with light sources as weak as an iPad screen. Toward optogenetic hearing restoration, ChReef enables stimulation of the auditory pathway in rodents and non-human primates with nanojoule thresholds, enabling efficient and frequency-specific stimulation by LED-based optical cochlear implants.

Abstract The role of the vertebrate retina in early vision is generally described by the efficient coding hypothesis 1,2 , which predicts that the retina reduces the redundancy inherent in natural scenes 3 by discarding spatiotemporal correlations while preserving stimulus information 4 . It is unclear, however, whether the predicted decorrelation and redundancy reduction in the activity of ganglion cells, the retina’s output neurons, hold under gaze shifts, which dominate the dynamics of the natural visual input 5 . We show here that species-specific gaze patterns in natural stimuli can drive correlated spiking responses both in and across distinct types of ganglion cells in marmoset as well as mouse retina. These concerted responses disrupt redundancy reduction to signal fixation periods with locally high spatial contrast. Model-based analyses of ganglion cell responses to natural stimuli show that the observed response correlations follow from nonlinear pooling of ganglion cell inputs. Our results indicate cell-type-specific deviations from efficient coding in retinal processing of natural gaze shifts.

Animals use visual objects to guide navigation-related behaviors. However, visual object-preferring areas have yet to be described in the mouse brain, limiting our understanding of how visual objects affect spatial navigation system processing. Using functional ultrasound imaging, we identified brain areas that were preferentially activated by images of objects compared with their scrambled versions. Whereas visual cortex did not show a preference, areas associated with spatial navigation were preferentially activated by visual objects. Electrophysiological recordings in postsubiculum, the cortical head direction (HD) system hub, confirmed a preference for visual objects in both HD cells and fast-spiking interneurons. In freely moving animals, visual objects increased firing rates of HD cells aligned with a visual object but decreased activity in HD cells coding for other directions.

Light-sheet microscopy is ideal for imaging large and cleared tissues, but achieving a high isotropic resolution for a centimeter-sized sample is limited by slow and often aberrated, axially scanned light sheets. Here, we introduce a compact, high-speed light-sheet fluorescence microscope achieving 850 nm isotropic resolution across cleared samples up to 1 cm³ and refractive indices ranging from 1.33 to 1.56. Using off-the-shelf optics, we combine an air objective and a meniscus lens with an axially swept light sheet to achieve diffraction-limited resolution and aberration correction. The effective field of view is increased by twofold by correcting the field curvature of the light sheet using a concave mirror in the remote focusing unit. Adapting the light sheet's motion with a closed-loop feedback enhances the imaging speed by tenfold, reaching 100 frames per second while maintaining resolution and field of view. We benchmark the system performance across scales, from subcellular structure up to centimeter scale, using various clearing methods.

Microbial rhodopsins are omnipresent on Earth; however, the vast majority of them remain uncharacterized. Here, we describe a rhodopsin group found in microorganisms from cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face. Combining single-particle cryo-electron microscopy and x-ray crystallography with rhodopsin activation by light, we demonstrate that the arginine stabilizes an ultraviolet (UV)-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the identified group. Our data suggest that CryoRs are sensors for UV irradiation and are also capable of inward proton translocation modulated by UV light.

Multi-scale X-ray phase contrast tomography (XPCT) enables three-dimensional (3D), non-destructive imaging of intact small animal cochlea and apical cochlear turns. Here we report on post-mortem imaging of excised non-human primate and rodent cochleae at different [Formula: see text]-CT and nano-CT synchrotron instruments. We explore different sample embeddings, stainings and imaging regimes. Under optimized conditions of sample preparation, instrumentation, imaging protocol, and phase retrieval, high image quality and detail level can be achieved in 3D reconstructions. The showcased instrumentation and imaging protocols along with the reconstucted volumes can serve as benchmarks and reference for multi-scale microanatomy and 3D histology. The provided benchmarks and imaging protocols of this work cover a wide range of scales and are intended as augmented imaging tools for auditory research.

Spatially nonlinear stimulus integration by retinal ganglion cells lies at the heart of various computations performed by the retina. It arises from the nonlinear transmission of signals that ganglion cells receive from bipolar cells, which thereby constitute functional subunits within a ganglion cell's receptive field. Inferring these subunits from recorded ganglion cell activity promises a new avenue for studying the functional architecture of the retina. This calls for efficient methods, which leave sufficient experimental time to leverage the acquired knowledge for further investigating identified subunits. Here, we combine concepts from super-resolution microscopy and computed tomography and introduce super-resolved tomographic reconstruction (STR) as a technique to efficiently stimulate and locate receptive field subunits. Simulations demonstrate that this approach can reliably identify subunits across a wide range of model variations, and application in recordings of primate parasol ganglion cells validates the experimental feasibility. STR can potentially reveal comprehensive subunit layouts within only a few tens of minutes of recording time, making it ideal for online analysis and closed-loop investigations of receptive field substructure in retina recordings.

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins or FARs) were identified and suggested to lack the retinal-binding pocket despite preserving the lysine residue in many members of the group. Here, we present cryoelectron microscopic (cryo-EM) structures of paralog FArhodopsin and proteorhodopsin from marine bacterium Pseudothioglobus, both forming pentamers similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of FArhodopsins.

ABSTRACT Background Optogenetics is a cutting‐edge approach that can enable direct stimulation of gastric smooth muscle cells (SMC) by combining cell‐specific overexpression of light‐sensitive proteins with light stimulation. We previously demonstrated that direct optogenetic stimulation of gastric SMC via depolarization can restore contractility and food propulsion and could become a new treatment strategy for gastroparesis. The human receptor Neuropsin (hOPN5) enables activation of G q signaling with UV light. Herein, we explore this new strategy for direct optogenetic stimulation of gastric SMC. Methods We used a transgenic mouse model expressing hOPN5 in fusion with eYFP. Antral longitudinal smooth muscle strips were used for isometric force measurements and whole stomachs for intragastric pressure measurements, comparing light stimulation to other stimuli. Adeno‐associated virus (AAV) serotypes were screened for efficiency in transducing cultured gastric SMC, and transduced cells were tested by Ca 2+ imaging. Results hOPN5 expression was restricted to and found in ~1/3 of SMC in the stomach. UV light induced isometric force and increased intragastric pressure only in transgenic mice similarly to electrical field stimulation and reached approximately 1/3 of the force induced by global depolarization and muscarinic receptor activation. Importantly, optical stimulation remained effective in an ex vivo gastroparesis model. AAV 2.5 was by far the most effective serotype for SMC transduction, and UV light triggered Ca 2+ transients in SMC expressing hOPN5. Conclusion hOPN5 is a new and effective tool to directly stimulate gastric SMC to control contractility with light. Thus, it is an additional and complementary approach to light‐induced membrane depolarization to restore gastric motility.

Abstract Objective. In case of deafness, electrical cochlear implants (eCIs) bypass dysfunctional or lost hair cells by direct stimulation of the auditory nerve. However, spectral selectivity of eCI sound coding is low as the wide current spread from each electrode activates large sets of neurons along the tonotopic axis of the cochlea. As light can be better confined in space, optical cochlear implants (oCIs) combined with cochlear optogenetics promise to overcome this shortcoming of eCIs. This requires appropriate sound processing and control of multiple microscale emitters. Approach. Here, we describe the development, characterisation, and application of a preclinical miniaturised low-weight and wireless LED-based multichannel oCI system for hearing restoration, and its comparison to its sister eCI system. We present exemplary implementation of these systems in behavioural studies on freely moving rats. Main results. The system, which weights 15 g, is 20 mm in diameter and 20 mm in height, performed for up to 8 h in behavioural experiments on freely moving rats proving its utility for cueing auditory tasks in deaf animals. Significance. The head-worn oCI system enabled deafened rats to perform a locomotion task in response to acoustic stimulation proving the concept of multichannel optogenetic hearing restoration in rodents. This paves the way for implementation in other species and development of future clinical oCI systems for improved hearing restoration.

Abstract Light sheet microscopy is the ideal technique for multiscale imaging of large and cleared tissues, and it is desirable to achieve the highest possible isotropic resolution across the entire sample. However, isotropic resolution for a centimeter-sized sample has only been achieved with slow and often aberrated, axially scanned light sheets, resulting in a low resolution of several micrometers. Here, we introduce a compact, high-speed light sheet fluorescence microscope with isotropic sub-micron resolution optimized for cleared tissue. We introduce three major opto-mechanical innovations using off-the-shelf optics to achieve an isotropic resolution of 850 nm across samples up to 1 cm 3 and refractive indices ranging from 1.33 to 1.56, using mechanical tiling with a field of view of 800 µm × 800 µm. We show that combining an air objective and a meniscus lens achieves an axially swept light sheet with sub-micron diffraction-limited resolution and aberration correction. The effective field of view is increased 2-fold by correcting the field curvature of the light sheet with a concave mirror in the remote focusing unit. Furthermore, the imaging speed is enhanced 10-fold by adapting the light sheet’s motion with a closed-loop feedback, reaching 100 frames per second while maintaining isotropic resolution across the large field of view. Finally, we showcase the performance of our light sheet system for imaging from subcellular up to centimeter scale in cleared zebrafish, mouse cochlea, and mouse brain using various clearing methods.

Gastroparesis is a disease in which gastric emptying is significantly decreased in the absence of a mechanical obstruction of the gastric outlet. In addition to conservative or pharmacological treatment options, gastric electrical stimulation (GES) is a possible alternative therapy for refractory courses of the disease. However, the available data from randomised controlled trials on efficacy do not permit an unqualified recommendation. The aim of this review is to introduce the clinical picture of gastroparesis, including the underlying pathophysiology, and to classify the therapeutic role of GES, based on the currently available literature. In particular, indications and limitations of the therapy are outlined that may lead to optimisation of the therapy, followed by an outlook on a potential new therapeutic procedure.

Abstract Activation of G protein coupled receptors coupling to the G i/o pathway leads to the activation of G protein-activated inward rectifier potassium channels (GIRK) in a fast membrane-delimited manner in excitable cells. Activation of GIRK causes the hyperpolarization of the cell membrane, where hyperpolarization is dependent on te availability of G i/o coupled GPCRs and GIRK. In particular, in optogenetic and chemogenetic experiments neuronal silencing depends on downstream targets of G i/o -coupled GPCRs. To selectively enhance G i/o mediated GIRK currents, we created expression cassettes consisting of a homomer forming GIRK subunit and various light-activated G i/o -coupled GPCRs (Melanopsin, Long-wave-sensitive opsin 1, Parapinopsin or Opsin 7b). We demonstrate that light-activation of the GIRK/GPCR constructs induces robust GIRK currents in human embryonic kidney 293 cells, cardiomyocytes and cerebellar Purkinje cells and changes the net effect of G protein signaling of the promiscuous Opn4L from a G q/11 mediated excitation towards an G i/o mediated inhibition. Thus, our tools enhance target selectivity and improve optogenetic control of the G i/o pathway by light in excitable cells.

Optogenetics allows versatile control of excitable cell networks, which advances basic science research and drives the development of future medical applications. Fast-closing channelrhodopsins (ChRs) are required for high temporal fidelity of neurostimulation, but their short channel open times require sufficient plasma membrane expression and high light intensity, challenging clinical translation. Here, we addressed the need of high-rate neurostimulation by engineering optimized blue-light-sensitive ChR variants. In particular, we report on the ChR2 variant f-ChR2 TC enabling high frequency stimulation at low light requirements, due to its good plasma membrane targeted expression and balanced closing kinetics. Upon Adeno-associated virus (AAV) mediated f-ChR2 TC expression in spiral ganglion neurons of the inner ear in mice, f-ChR2 TC accordingly enabled optogenetic stimulation of the auditory nerve with sizeable responses beyond 300 Hz and low pulse energy thresholds. Translating the approach to the larger cochlea of gerbils, we tested the utility of f-ChR2 TC for evaluating multichannel optical cochlear implants with blue light emitting diodes and found light-efficient stimulation of the auditory pathway by single LEDs at rates ≥100 Hz.

Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein synaptotagmin-1. Cryo-ET imaging at sites of colocalization predominantly revealed excitatory synapses. Our method allows structural studies of synapse-resident protein complexes in situ, facilitating investigations of the molecular architecture of synapses.

The dichotomy of excitation and suppression is one of the canonical mechanisms explaining the complexity of neural activity. Computational models of the interplay of excitation and suppression in single neurons aim at investigating how this interaction affects a neuron's spiking responses and shapes the encoding of sensory stimuli. Here, we compare the performance of three filter-based stimulus-encoding models for predicting retinal ganglion cell responses recorded from axolotl, mouse, and marmoset retina to different types of temporally varying visual stimuli. Suppression in these models is implemented via subtractive or divisive interactions of stimulus filters or by a response-driven feedback module. For the majority of ganglion cells, the subtractive and divisive models perform similarly and outperform the feedback model as well as a linear-nonlinear (LN) model with no suppression. Comparison between the subtractive and the divisive model depends on cell type, species, and stimulus components, with the divisive model generalizing best across temporal stimulus frequencies and visual contrast and the subtractive model capturing in particular responses for slow temporal stimulus dynamics and for slow axolotl cells. Overall, we conclude that the divisive and subtractive models are well suited for capturing interactions of excitation and suppression in ganglion cells and perform best for different temporal regimes of these interactions.

Efficient detection of breathing impairment is critical for treatment and prognosis in neuromuscular disorders. However, standard pulmonary function tests often yield ambiguous results. This prospective study evaluates whether advanced real-time MRI (RT-MRI) combined with deep learning-based image segmentation provides sensitive outcome measures for respiratory dysfunction in late-onset Pompe disease (LOPD), a model disease for diaphragmatic weakness. Eleven Pompe patients (mean age 52.2 years; 55% female) and 11 controls (mean age 50.9 years; 55% female) were included. RT-MRI with a temporal resolution of 50 ms, combined with U-Net-supported lung segmentation, revealed significantly reduced diaphragmatic motion in Pompe patients compared to controls and unmasked paradoxical diaphragmatic motion in Pompe patients (7 of 11). Reduced diaphragmatic sniff velocity and pathological diaphragmatic/thoracic synchronicity were detected in Pompe patients with still normal results in standard pulmonary function tests. Fatty involution of the diaphragm as quantified by fast T1 mapping correlated significantly with functional parameters from RT-MRI and pulmonary function tests. RT-MRI combined with deep learning-based lung segmentation offers novel biomarkers for early detection of respiratory muscle weakness. This new technique provides useful outcome measures for clinical care as well as treatment studies in patients with neuromuscular breathing impairment. The technique can also be used to characterize physiologic breathing patterns in healthy individuals.

BACKGROUND: Swallowing dysfunction-dysphagia-is a frequent and debilitating symptom in neuromuscular disorders, leading to malnutrition, cachexia, aspiration pneumonia, and death. Identification of the underlying pathophysiological mechanisms is important for diagnosis and treatment. As standard assessments have limitations, novel imaging techniques are needed. We here studied the utility of real-time MRI and quantitative muscle ultrasound for characterizing dysphagia in two different neuromuscular disorders. METHODS: This prospective cohort study included 18 patients with inclusion body myositis (IBM, 33% female, age 68.9 ± 7.7 years) and 13 with oculopharyngeal muscular dystrophy (OPMD, 62% female, age 55.9 ± 7.0 years) from two European Neuromuscular research centers (Nijmegen, NL; Göttingen, DE). Swallowing function was studied using real-time MRI (RT-MRI), FEES (flexible endoscopic evaluation of swallowing), and clinical assessments. T1-mapping and quantitative muscle ultrasound (QMUS) were used to analyse tissue properties in swallowing muscles. Outcomes were compared between the two muscle diseases. RT-MRI values were also compared with 22 age- and sex-matched non-myopathic controls. RESULTS: RT-MRI revealed significantly prolonged oral transit times in OPMD vs. controls (difference between means = 581.2 ms, 95% CI 225.9-936.4, p = 0.002). Pharyngeal transit time was significantly prolonged in IBM vs. controls (difference between means = 1132.8 ms, 95% CI 482.2-1783, p = 0.001). A cricopharyngeal bar as a well-established morphological indicator of dysphagia was identified in 80% of patients with IBM compared with 53% in OPMD. Fatty degeneration of the tongue in OPMD significantly correlated between MRI-T1 values and ultrasound echogenicity (Spearman's ρ = -0.52, p = 0.005). ROC revealed excellent discrimination between diseases by combining RT-MRI, T1-mapping and QMUS (AUC = 0.95, 95% CI 0.86-1.00), while FEES and clinical assessments failed to differentiate specific patterns of dysphagia. CONCLUSIONS: This study supports the value of novel MRI and ultrasound techniques for clinical use by identifying the pathophysiology and severity of impaired swallowing. Differentiating the phenotypes of dysphagia can aid in the diagnosis and treatment of affected patients. RT-MRI and QMUS may serve as outcome measures for swallowing in clinical trials.

Disabling hearing impairment is a common human sensory deficit. OTOF is a major deafness gene. It codes for the synaptic protein otoferlin and is essential for transmitter release by inner hair cells (IHCs). Upon genetic loss of otoferlin, cochlear structure and function remain intact up to the IHC synapses, which fail to encode sound. Building on preclinical hearing restoration by AAV-mediated cochlear gene transfer in mice, clinical OTOF-gene-therapy trials are now targeting the pediatric population. However, preclinical optimization and characterization remain urgent needs for the development of OTOF-gene-therapy. Here, we report on the generation and characterization of a marmoset KO that models OTOF-related auditory synaptopathy and can thus address these needs. Following ovary stimulation, harvesting, in vitro maturation and fertilization of oocytes, we injected the zygotes with Cas9 and guide RNAs to disrupt OTOF. Mutant embryos were transferred into the uterus of foster mothers. Marmosets with biallelic, non-mosaic OTOF-KO were normally born and raised by their respective foster parents. Auditory brainstem recordings and otoacoustic emissions revealed profound auditory synaptopathy and OTOF-KO was further validated by the lack of otoferlin expression in IHCs. The new non-human primate model of OTOF-related auditory synaptopathy will serve studies of specificity, efficacy, and longevity of novel inner ear therapies.

Abstract Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow robust synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein Synaptotagmin-1. Cryo-ET imaging at sites of colocalization consistently revealed excitatory synapses. Our method allows structural studies of synaptic protein complexes in situ , facilitating investigations of the molecular architecture of synapses.