Feasibility of Endovascular Deep Brain Stimulation of Anterior Nucleus of the Thalamus for Refractory Epilepsy.

BACKGROUND AND OBJECTIVES: Deep brain stimulation (DBS) has developed into an effective therapy for several disease states including treatment-resistant Parkinson disease and medically intractable essential tremor, as well as segmental, generalized and cervical dystonia, and obsessive-compulsive disorder (OCD). Dystonia and OCD are approved with Humanitarian Device Exemption. In addition, DBS is also approved for the treatment of epilepsy in the anterior nucleus of the thalamus. Although overall considered an effective treatment for Parkinson disease and epilepsy, a number of specific factors determine the treatment success for DBS including careful patient selection, effective postoperative programming of DBS devices and accurate electrode placement. Furthermore, invasiveness of the procedure is a rate limiter for patient adoption. It is desired to explore a less invasive way to deliver DBS therapy. METHODS: Here, we report for the first time the direct comparison of endovascular and parenchymal DBS in a triplicate ovine model using the anterior nucleus of the thalamus as the parenchymal target for refractory epilepsy. RESULTS: Triplicate ovine studies show comparable sensing resolution and stimulation performance of endovascular DBS with parenchymal DBS. CONCLUSION: The results from this feasibility study opens up a new frontier for minimally invasive DBS therapy.

Personalized inference for neurostimulation with meta-learning: a case study of vagus nerve stimulation.

Objective. Neurostimulation is emerging as treatment for several diseases of the brain and peripheral organs. Due to variability arising from placement of stimulation devices, underlying neuroanatomy and physiological responses to stimulation, it is essential that neurostimulation protocols are personalized to maximize efficacy and safety. Building such personalized protocols would benefit from accumulated information in increasingly large datasets of other individuals' responses.Approach. To address that need, we propose a meta-learning family of algorithms to conduct few-shot optimization of key fitting parameters of physiological and neural responses in new individuals. While our method is agnostic to neurostimulation setting, here we demonstrate its effectiveness on the problem of physiological modeling of fiber recruitment during vagus nerve stimulation (VNS). Using data from acute VNS experiments, the mapping between amplitudes of stimulus-evoked compound action potentials (eCAPs) and physiological responses, such as heart rate and breathing interval modulation, is inferred.Main results. Using additional synthetic data sets to complement experimental results, we demonstrate that our meta-learning framework is capable of directly modeling the physiology-eCAP relationship for individual subjects with much fewer individually queried data points than standard methods.Significance. Our meta-learning framework is general and can be adapted to many input-response neurostimulation mapping problems. Moreover, this method leverages information from growing data sets of past patients, as a treatment is deployed. It can also be combined with several model types, including regression, Gaussian processes with Bayesian optimization, and beyond.

Modulating functionally-distinct vagus nerve fibers using microelectrodes and kilohertz frequency electrical stimulation.

Modulation of functionally distinct nerve fibers with bioelectronic devices provides a therapeutic opportunity for various diseases. In this study, we began by developing a computational model including four major subtypes of myelinated fibers and one unmyelinated fiber. Second, we used an intrafascicular electrode to perform kHz-frequency electric stimulation to preferentially modulate a population of fibers. Our model suggests that fiber physical properties and electrode-to-fascicle distance severely impacts stimulus-response relationships. Large diameter fibers (Aα- and Aß-) were only minimally influenced by the fascicle size and electrode location, while smaller diameter fibers (Aδ-, B- and C-) indicated a stronger dependency.Clinical Relevance- Our findings support the possibility of selectively modulating functionally-distinct nerve fibers using electrical stimulation in a small, localized region. Our model provides an effective tool to design next-generation implantable devices and therapeutic stimulation strategies toward minimizing off-target effects.

Organ- and function-specific anatomical organization of vagal fibers supports fascicular vagus nerve stimulation.

Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Existing vagus nerve stimulation (VNS) therapies activate vagal fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. The transverse and longitudinal arrangement of fibers inside the vagal trunk with respect to the functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using micro-computed tomography imaging, we tracked fascicular trajectories and found that, in swine, sensory and motor fascicles are spatially separated cephalad, close to the nodose ganglion, and merge caudad, towards the lower cervical and upper thoracic region; larynx-, heart- and lung-specific fascicles are separated caudad and progressively merge cephalad. Using quantified immunohistochemistry at single fiber level, we identified and characterized all vagal fibers and found that fibers of different morphological types are differentially distributed in fascicles: myelinated afferents and efferents occupy separate fascicles, myelinated and unmyelinated efferents also occupy separate fascicles, and small unmyelinated afferents are widely distributed within most fascicles. We developed a multi-contact cuff electrode to accommodate the fascicular structure of the vagal trunk and used it to deliver fascicle-selective cervical VNS in anesthetized and awake swine. Compound action potentials from distinct fiber types, and physiological responses from different organs, including laryngeal muscle, cough, breathing, and heart rate responses are elicited in a radially asymmetric manner, with consistent angular separations that agree with the documented fascicular organization. These results indicate that fibers in the trunk of the vagus nerve are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.

Understanding the effect of social media marketing activity for promoting intention to participate in martial arts.

The development of folk martial arts in China has encountered many obstacles and difficulties in promoting the sport. Although there are many martial arts-related groups on WeChat, the largest social media in China, the interaction is not enthusiastic enough and the participation is too low. The main purpose of this study is to understand the impact of social media marketing activities and user experience on the intention of people to participate in martial arts through a quantitative research method. After the literature study, a research model was developed based on Theory of Planned Behavior (TPB), in which the constructs include social media marketing activities, user experience, attitudes toward martial arts, subjective norms, martial arts attachment, and participation intention. The results of the study illustrated that social media marketing activities and user experience had a positive and significant effect on martial arts attitudes, subjective norms, and martial arts attachment via Structural Equation Modeling (SEM). Martial arts attitudes, subjective norms, and martial arts attachment had a positive and significant effect on the intention to participate. Finally, based on the results of this study, we propose suggestions for social media marketing activities, user experience, martial arts attachment, attitudes toward martial arts, subjective norms, and martial arts participation intentions for martial arts social media operators, martial arts promotion organizations, and subsequent studies.

kHz-frequency electrical stimulation selectively activates small, unmyelinated vagus afferents.

BACKGROUND: Vagal reflexes regulate homeostasis in visceral organs and systems through afferent and efferent neurons and nerve fibers. Small, unmyelinated, C-type afferents comprise over 80% of fibers in the vagus and form the sensory arc of autonomic reflexes of the gut, lungs, heart and vessels and the immune system. Selective bioelectronic activation of C-afferents could be used to mechanistically study and treat diseases of peripheral organs in which vagal reflexes are involved, but it has not been achieved. METHODS: We stimulated the vagus in rats and mice using trains of kHz-frequency stimuli. Stimulation effects were assessed using neuronal c-Fos expression, physiological and nerve fiber responses, optogenetic and computational methods. RESULTS: Intermittent kHz stimulation for 30 min activates specific motor and, preferentially, sensory vagus neurons in the brainstem. At sufficiently high frequencies (>5 kHz) and at intensities within a specific range (7-10 times activation threshold, T, in rats; 15-25 × T in mice), C-afferents are activated, whereas larger, A- and B-fibers, are blocked. This was determined by measuring fiber-specific acute physiological responses to kHz stimulus trains, and by assessing fiber excitability around kHz stimulus trains through compound action potentials evoked by probing pulses. Aspects of selective activation of C-afferents are explained in computational models of nerve fibers by how fiber size and myelin shape the response of sodium channels to kHz-frequency stimuli. CONCLUSION: kHz stimulation is a neuromodulation strategy to robustly and selectively activate vagal C-afferents implicated in physiological homeostasis and disease, over larger vagal fibers.

Strategies for precision vagus neuromodulation.

The vagus nerve is involved in the autonomic regulation of physiological homeostasis, through vast innervation of cervical, thoracic and abdominal visceral organs. Stimulation of the vagus with bioelectronic devices represents a therapeutic opportunity for several disorders implicating the autonomic nervous system and affecting different organs. During clinical translation, vagus stimulation therapies may benefit from a precision medicine approach, in which stimulation accommodates individual variability due to nerve anatomy, nerve-electrode interface or disease state and aims at eliciting therapeutic effects in targeted organs, while minimally affecting non-targeted organs. In this review, we discuss the anatomical and physiological basis for precision neuromodulation of the vagus at the level of nerve fibers, fascicles, branches and innervated organs. We then discuss different strategies for precision vagus neuromodulation, including fascicle- or fiber-selective cervical vagus nerve stimulation, stimulation of vagal branches near the end-organs, and ultrasound stimulation of vagus terminals at the end-organs themselves. Finally, we summarize targets for vagus neuromodulation in neurological, cardiovascular and gastrointestinal disorders and suggest potential precision neuromodulation strategies that could form the basis for effective and safe therapies.

Implant- and anesthesia-related factors affecting cardiopulmonary threshold intensities for vagus nerve stimulation.

Objective.Vagus nerve stimulation (VNS) is typically delivered at increasing stimulus intensity until a neurological or physiological response is observed ('threshold') for dose calibration, preclinically and therapeutically. Factors affecting VNS thresholds have not been studied systematically. In a rodent model of VNS we measured neural and physiological responses to increasing VNS intensity, determined neurological and physiological thresholds and examined the effect of implant- and anesthesia-related factors on thresholds.Approach.In acute and chronic vagus implants (45 and 20 rats, respectively) VNS was delivered under isoflurane, ketamine-xylazine, or awake conditions. Evoked compound action potentials (CAPs) were recorded and activation of different fiber types was extracted. Elicited physiological responses were registered, including changes in heart rate (HR), breathing rate (BR), and blood pressure (BP). CAP and physiological thresholds were determined.Main results. The threshold for evoking discernable CAPs (>10µV) (CAP threshold) is significantly lower than what elicits 5%-10% drop in heart rate (heart rate threshold, HRT) (25µA ± 1.8 vs. 80µA ± 5.1, respectively; mean ± SEM). Changes in BP and small changes in BR (bradypnea) occur at lowest intensities (70µA ± 8.3), followed by HR changes (80µA ± 5.1) and finally significant changes in BR (apnea) (310µA ± 32.5). HRT and electrode impedance are correlated in chronic (Pearson correlationr= 0.47;p< 0.001) but not in acute implants (r= -0.34;pNS); HRT and impedance both increase with implant age (r= 0.44;p< 0.001 andr= 0.64;p< 0.001, respectively). HRT is lowest when animals are awake (200µA ± 35.5), followed by ketamine-xylazine (640µA ± 151.5), and isoflurane (1000µA ± 139.5). The sequence of physiological responses with increasing VNS intensity is the same in anesthetized and awake animals. Pulsing frequency affects physiological responses but not CAPs.Significance. Implant age, electrode impedance, and type of anesthesia affect VNS thresholds and should be accounted for when calibrating stimulation dose.

Development and characterization of a chronic implant mouse model for vagus nerve stimulation.

Vagus nerve stimulation (VNS) suppresses inflammation and autoimmune diseases in preclinical and clinical studies. The underlying molecular, neurological, and anatomical mechanisms have been well characterized using acute electrophysiological stimulation of the vagus. However, there are several unanswered mechanistic questions about the effects of chronic VNS, which require solving numerous technical challenges for a long-term interface with the vagus in mice. Here, we describe a scalable model for long-term VNS in mice developed and validated in four research laboratories. We observed significant heart rate responses for at least 4 weeks in 60-90% of animals. Device implantation did not impair vagus-mediated reflexes. VNS using this implant significantly suppressed TNF levels in endotoxemia. Histological examination of implanted nerves revealed fibrotic encapsulation without axonal pathology. This model may be useful to study the physiology of the vagus and provides a tool to systematically investigate long-term VNS as therapy for chronic diseases modeled in mice.

A Computational Model of Functionally-distinct Cervical Vagus Nerve Fibers.

Cervical vagus nerve stimulation (VNS) is a neuromodulation therapy used in the treatment of several chronic disorders. In order to maximize the therapeutic effectiveness of VNS, it has become increasingly important to deliver fiber-specific neurostimulation, so that undesired effects can be minimized. Assessing the activation of different vagal fiber types through electrical stimulation is therefore essential for developing fiber-selective VNS therapies. Towards this goal, we conducted in silico investigations using a generic model of functionally distinct nerve fibers and clinically relevant cuff electrodes using COMSOL. Our model is constrained by histological observations from rat cervical vagus nerves and its outputs are validated against averaged compound nerve action potentials (CNAPs) obtained from rat vagus nerve recordings. We propose this model as an effective tool to design fiber-specific stimulation protocols before testing them in experimental animals.

Quantitative estimation of nerve fiber engagement by vagus nerve stimulation using physiological markers.

BACKGROUND: Cervical vagus nerve stimulation (VNS) is an emerging bioelectronic treatment for brain, metabolic, cardiovascular and immune disorders. Its desired and off-target effects are mediated by different nerve fiber populations and knowledge of their engagement could guide calibration and monitoring of VNS therapies. OBJECTIVE: Stimulus-evoked compound action potentials (eCAPs) directly provide fiber engagement information but are currently not feasible in humans. A method to estimate fiber engagement through common, noninvasive physiological readouts could be used in place of eCAP measurements. METHODS: In anesthetized rats, we recorded eCAPs while registering acute physiological response markers to VNS: cervical electromyography (EMG), changes in heart rate (ΔHR) and breathing interval (ΔBI). Quantitative models were established to capture the relationship between A-, B- and C-fiber type activation and those markers, and to quantitatively estimate fiber activation from physiological markers and stimulation parameters. RESULTS: In bivariate analyses, we found that EMG correlates with A-fiber, ΔHR with B-fiber and ΔBI with C-fiber activation, in agreement with known physiological functions of the vagus. We compiled multivariate models for quantitative estimation of fiber engagement from these markers and stimulation parameters. Finally, we compiled frequency gain models that allow estimation of fiber engagement at a wide range of VNS frequencies. Our models, after calibration in humans, could provide noninvasive estimation of fiber engagement in current and future therapeutic applications of VNS.

Anodal block permits directional vagus nerve stimulation.

Vagus nerve stimulation (VNS) is a bioelectronic therapy for disorders of the brain and peripheral organs, and a tool to study the physiology of autonomic circuits. Selective activation of afferent or efferent vagal fibers can maximize efficacy and minimize off-target effects of VNS. Anodal block (ABL) has been used to achieve directional fiber activation in nerve stimulation. However, evidence for directional VNS with ABL has been scarce and inconsistent, and it is unknown whether ABL permits directional fiber activation with respect to functional effects of VNS. Through a series of vagotomies, we established physiological markers for afferent and efferent fiber activation by VNS: stimulus-elicited change in breathing rate (ΔBR) and heart rate (ΔHR), respectively. Bipolar VNS trains of both polarities elicited mixed ΔHR and ΔBR responses. Cathode cephalad polarity caused an afferent pattern of responses (relatively stronger ΔBR) whereas cathode caudad caused an efferent pattern (stronger ΔHR). Additionally, left VNS elicited a greater afferent and right VNS a greater efferent response. By analyzing stimulus-evoked compound nerve potentials, we confirmed that such polarity differences in functional responses to VNS can be explained by ABL of A- and B-fiber activation. We conclude that ABL is a mechanism that can be leveraged for directional VNS.

An impedance matching algorithm for common-mode interference removal in vagus nerve recordings.

BACKGROUND: The peripheral nervous system is involved in a multitude of physiological functions. Recording neural signals provides information that can be used by diagnostic bioelectronic medicine devices, closed-loop neuromodulation therapies and other neuroprosthetic applications. The ability to accurately record these signals is challenging, due to the presence of various biological and instrument-related interference sources. NEW METHOD: We developed a common-mode interference rejection algorithm based on an impedance matching approach for bipolar cuff electrodes. Two unipolar channels were recorded from the two electrode contacts of a bipolar cuff. The impedance mismatch was estimated and used to correct one of the two channels. RESULTS: When applied to electrocardiographic (ECG) artifacts collected from three mice using CorTec electrodes, the algorithm reduced the interference to noise ratio (INR) over simple subtraction by 12 dB on average. The algorithm also reduced the INR of stimulation artifacts in recordings from three rats collected using flexible electrodes by an additional 2.4 dB. In the same experiments evoked electromyographic (EMG) interference was suppressed by 1.3 dB. COMPARISON WITH EXISTING METHODS: Simple subtraction is the common approach for reducing common-mode interference in bipolar recordings, however impedance mismatches that exist or emerge compromise its efficiency. CONCLUSIONS: The algorithm significantly reduced the common-mode interference from ECG artifacts, stimulation artifacts, and evoked EMG interference, while retaining neural signals, in two animal models and two recording setups. This approach can be used in a variety of different neurophysiological setups to remove common-mode interference from a variety of sources.

Extraction of Evoked Compound Nerve Action Potentials from Vagus Nerve Recordings.

Cervical vagus nerve stimulation (VNS) is a neuromodulation therapy for the treatment of several chronic disorders. The effects of VNS are mediated by activation of nerve fibers of different types. In order to maximize the desired and minimize the undesired effects of VNS, assessing activation of vagal fiber types by VNS is essential. Evoked compound nerve action potentials (CNAPs) are commonly used as a method to estimate vagal fiber activation in the context of neurostimulation. However, vagal CNAPs are frequently contaminated by signals from non-neural sources, like electrocardiography (ECG), stimulus artifacts and evoked electromyographic (EMG) activity. In this study, we present a systematic methodology for suppressing non-neural signals in CNAP recordings from the rat vagus. The methodology involves intravenous infusion of vecuronium under ventilation, for suppressing EMG, and digital and analog signal processing, for suppressing ECG and stimulus artifacts, respectively. We compiled A-, B- and C-type fiber activation profiles with and without this methodology and found that our method significantly increased the reliability of CNAPs. We found that the A-component is obscured by the stimulus artifact, whereas the B- and C-components are frequently contaminated by evoked EMG. We extracted CNAPs evoked by square pulses of different polarities and amplitudes and documented effects consistent with well-established biophysical attributes of VNS.

Stimulation strategies for selective activation of retinal ganglion cell soma and threshold reduction.

OBJECTIVE: Retinal prosthetic implants restore partial vision to patients blinded due to outer retinal degeneration, using a camera-guided multielectrode array (MEA) that electrically stimulates surviving retinal neurons. Commercial epi-retinal prostheses use millisecond-scale charge-balanced, symmetric, cathodic-first biphasic pulses to depolarize retinal ganglion cells (RGCs) and bipolar cells (BCs), frequently creating oblong perceptions of light related to axonal activation of RGCs. Stimulation strategies that avoid axonal stimulation and decrease the threshold of targeted neurons may significantly improve prosthetic vision in terms of spatial resolution and power efficiency. APPROACH: We developed a virus-transduced genetically encoded calcium indicator (GECI) GCaMP6f and microscopy platform for calcium imaging to record the neural activity from RGCs at single-cell resolution in wholemount retinas. Multiple stimulation paradigms were applied through a microelectrode array (MEA) with transparent indium tin oxide electrodes. The evoked neuronal activities were converted to corresponding 2D calcium imaging transient pattern and spatial threshold map to identify the ideal focal response which corresponds to optimal percept in patient. MAIN RESULTS: The proposed optical system with GCaMP6f is capable of recording from population of mouse RGCs in real time during electrical stimulation with precise location information relative to the stimulation sites. Optimal duration and phase order of pulse were identified to avoid axonal stimulation and selectively activate targeted RGC somas, without requiring a significant increase in stimulation charge. Additionally, we show that reduced stimulus threshold can be achieved with the special design of asymmetric anodic-first pulse. SIGNIFICANCE: Our findings support the possibility of manipulating the responses of RGCs through varying the stimulation waveform. Focal response can be achieved with relative short duration (⩽120 µs) pulses, and can be improved by reversing the standard phase order. The RGCs threshold can be significantly reduced by 33.3%-50% in terms of charge through applying hyperpolarizing pre-pulses with a 20:1 ratio (pre-pulse:stimulus pulse). The results support the future retinal prosthesis design that potentially forms more ideal shape perception with higher spatial resolution and power efficiency.

GCaMP expression in retinal ganglion cells characterized using a low-cost fundus imaging system.

OBJECTIVE: Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable. APPROACH: We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation. MAIN RESULTS: The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation. SIGNIFICANCE: The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.

Method to remove photoreceptors from whole mount retina in vitro.

Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input.NEW & NOTEWORTHY This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording.

Stimulation Strategies for Selective Activation of Retinal Ganglion Cells.

Retinal prosthetic implants have shown potential to restore partial vision to patients blinded by retinitis pigmentosa or dry age-related macular degeneration, via a camera-driven multielectrode array that electrically stimulates surviving retinal neurons. Commercial epi-retinal prostheses mostly use charge-balanced symmetric cathodic-first biphasic pulses to depolarize retinal ganglion cells (RGCs) and bipolar cells (BCs), resulting in the perception of light in blind patients. However, previous clinical study for patients with Argus II epiretinal implants reported most percepts evoked by single electrode stimulation were elongated and aligned with estimated axon path of retinal ganglion cells, suggesting the activation of axon bundles. In this project, using an established genetically encoded calcium indicator (GECI), we performed in vitro calcium imaging for different stimulation paradigms, focusing primarily on short duration pulse that can avoid axonal stimulation and selective activate targeted RGC soma. The findings support the possibility to manipulate the responses of RGCs through varying the stimulation waveform, thus potentially forming more ideal shape perception with higher spatial resolution in future retinal prosthesis design.

In vivo characterization of genetic expression of virus-transduced calcium indicators in retinal ganglion cells using a low-cost funduscope.

Virus-transduced calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. To track the level of in vivo expression of genetically encoded calcium indicators (GECIs) in rodent retina, we developed a noninvasive imaging approach based on a custom-modified low-cost and simple fundus system that enabled us to monitor and characterize in vivo bright-field and fluorescence retinal image. The system clearly resolves individual retinal ganglion cells (RGCs) and axons. RGC fluorescence intensity and number of observable fluorescent cells show a consistent rising trend from week 1 to week 3 after viral injection, indicating a uniform increase of GCaMP6f expression. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array (MEA) and used calcium imaging to identify the optimal time for studying the responsiveness of RGCs to external electrical stimulation. The results show that the fluorescence-endoscopy fundus system is a powerful and widely accessible tool for evaluating in vivo fluorescence reporter expression.