Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

A digitális fonokardiográfia szerepe a kutyák szívhangjainak hallgatózásos vizsgálatának oktatásában.

Absztrakt Sok állatorvostanhallgatónak esik nehezére a szívhangok helyes értelmezése. Kutatásunk célja a digitális fonokardiogramokat felhasználó online, tanterven kívüli oktatás hatásának felmérése volt a hallgatók szívhangok felismerésével kapcsolatos diagnosztikai képességeire. Harminchat véletlenszeruen elosztott állatorvostanhallgató hallgatott meg és értelmezett 10 hangfelvételt élettani szívhangokról (2), szívzörejekrol (4) és szívritmuszavarokról (4) egy négyhetes periódus elején és Végén. Huszonkét hallgató vett részt DPCG-ket felhasználó képzésben, egy önképzo oldal (n = 12) és egy online webinar (n = 10) keretein belül. A két teszten elért eredményeiket egy kontrollcsoportéval vetettük össze (n = 14), akik nem vettek részt tanterven kívüli képzésben. Bár az elo- és utótesztek pontszámai nem különböztek szignifikánsan a csoportok között, mindkét oktatásban résztvevo csoport szignifikáns csoporton belüli javulást mutatott a két teszt között (p = 0,023, p = 0,037), míg a kontrollcsoport nem (p = 0,49). Bár egyik csoport sem ért el javulást az élettani szívhangok felismerésében és szívritmuszavaroktól való elkülönítésében, mindkét csoportban javulást láttunk a szívzörejek felismerésében és azonosításában, valamint az észlelt szívritmuszavarok azonosításában. Eredményeink alapján a hallgatók hallgatózásos vizsgálattal kapcsolatos diagnosztikai képességei javíthatóak tanterven kívüli oktatással, akár élo álatok igénybevétele nélkül is. További, nagyobb elemszámmal végzett, valamint DPCG-t nem használó oktatómódszereket alkalmazó kutatás szükséges a DPCG-k szerepének pontos meghatározásához. Egy emelt elemszámmal végzett kutatás lehetové tenné továbbá egy kombinált oktatott csoport létrehozását, mérhetové téve a két módszer kumulatív hatását. This translation was provided by the authors. To view the original article visit: https://doi.org/10.3138/jvme-2021-0097.

Remote Teaching of Canine Cardiac Auscultation Using Digital Phonocardiograms.

Veterinary students often struggle to correctly interpret heart sounds. This study sought to evaluate if additional online training using digital phonocardiograms (DPCGs) improves students' ability to identify normal and pathologic heart sounds in dogs. Thirty-six randomly assigned veterinary students listened to and interpreted 10 audio recordings of normal heart sounds (2), heart murmurs (4), and arrhythmias (4) at the start and the end of a 4-week period. Twenty-two students participated in training with DPCGs, including those created from these recordings during this period, via a self-study website (n = 12) or online webinar (n = 10). Their results were compared with those of a control group (n = 14) that did not undergo additional training. Although pre- and post-training test scores did not differ between groups, both training groups showed within-group improvement between the two tests (p = .024, p = .037); the control group did not (p = .49). Although neither training group showed differences in ability to differentiate normal heart sounds from arrhythmias, both showed increased ability to detect and specify heart murmurs and provide refined diagnoses of detected arrhythmias. These results suggest additional training, even without actual patients, improves students' ability to identify heart murmurs and provide specific diagnoses for arrhythmias. Further study with a larger sample size and an additional group without DPCG-based training would help evaluate the effectiveness of DPCGs regarding arrhythmias. Studying a larger sample size would also allow for a training group participating in both training methods, measuring cumulative effectiveness of both methods.

Transience of the Retinal Output Is Determined by a Great Variety of Circuit Elements.

Retinal ganglion cells (RGCs) encrypt stimulus features of the visual scene in action potentials and convey them toward higher visual centers in the brain. Although there are many visual features to encode, our recent understanding is that the ~46 different functional subtypes of RGCs in the retina share this task. In this scheme, each RGC subtype establishes a separate, parallel signaling route for a specific visual feature (e.g., contrast, the direction of motion, luminosity), through which information is conveyed. The efficiency of encoding depends on several factors, including signal strength, adaptational levels, and the actual efficacy of the underlying retinal microcircuits. Upon collecting inputs across their respective receptive field, RGCs perform further analysis (e.g., summation, subtraction, weighting) before they generate the final output spike train, which itself is characterized by multiple different features, such as the number of spikes, the inter-spike intervals, response delay, and the rundown time (transience) of the response. These specific kinetic features are essential for target postsynaptic neurons in the brain in order to effectively decode and interpret signals, thereby forming visual perception. We review recent knowledge regarding circuit elements of the mammalian retina that participate in shaping RGC response transience for optimal visual signaling.

Digital phonocardiography of cardiac arrhythmias in dogs - Preliminary experiences.

Electronic stethoscopes and digital phonocardiograms (DPCGs) can be applied when diagnosing cardiac murmurs, but their use for cardiac arrhythmias is not described in veterinary medicine. Data of 10 dogs are presented in this preliminary study, demonstrating the applicability of these techniques. Although the number of artefacts and the amount of baseline noise produced by the two digitising systems used did not differ, the Welch Allyn Meditron system or similar ones capable of simultaneous recording of electrocardiograms (ECGs) and DPCGs provide a better option for clinical research and education, whilst the 3M Littmann 3200 system might be more suitable for everyday clinical settings. A combined system with simultaneous phonocardiogram and ECG, especially with wireless transmission, might be a solution in the future.

The role of gap junctions in cell death and neuromodulation in the retina.

Vision altering diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, myopia, retinal vascular disease, traumatic brain injuries and others cripple many lives and are projected to continue to cause anguish in the foreseeable future. Gap junctions serve as an emerging target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells, thereby playing a crucial role in pathophysiology. Since they are permeable for macromolecules, able to cross the cellular barriers, they show duality in illness as a cause and as a therapeutic target. In this review, we take recent advancements in gap junction neuromodulation (pharmacological blockade, gene therapy, electrical and light stimulation) into account, to show the gap junction's role in neuronal cell death and the possible routes of rescuing neuronal and glial cells in the retina succeeding illness or injury.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Teaching Tip: Virtual Oncology Clinic.

Due to limitations in traditional approaches to didactic and clinical learning, professional veterinary medical students face challenges in developing skills and competencies related to clinical practice. The Veterinary Information Network's (VIN) Virtual Clinic (VVC) aims to support learning by using gaming techniques to simulate clinical case management in a low-risk setting. The VVC lets students explore medical scenarios inside a virtual hospital. The purpose of this article is to describe the development and implementation of a learning approach that blends classroom instructor-directed learning with online simulation-based learning, using the VVC. We share challenges and successes of this approach. The case vignettes in the specific example described herein are for canine multicentric lymphoma. However, the lessons learned through the implementation of this oncology clinic module are expected to apply to a wide range of clinical disciplines.

Establishment of methodology for non-invasive electrocardiographic measurements in turtles and tortoises.

The lack of knowledge on how to perform species-specific electrocardiographic (ECG) measurements in chelonians makes ECG evaluations difficult. The aim of this study was to establish non-invasive methods for ECG sample collection in different species of non-anaesthetised chelonians, focusing on adhesive and clamp electrodes. A total of 72 turtles and tortoises from 20 species and various sizes were used for the study. Body weight ranged from 32 g to 65 kg. From the aspect of specimen fixation, dorsal recumbency proved to be the most useful. Both adhesive and clamp electrodes yielded results when applied to the plastron and skin folds. Pre-emptive results suggest an indirect correlation with plastron thickness, the presence of a hinge, habitat and measurable ECG wave amplitude. ECG wave recordings are more likely in aquatic chelonians and species with a hinge. With size the plastron also thickens, making wave detection impossible. ECG waves were detected in 41 of the 72 specimens, complete PQRST complexes in 19 animals, with the rest showing absent P waves in all leads. ECG amplitudes were below 1 mV, with an average of 0.15 mV R wave amplitude.

Review of gastric torsion in eight guinea pigs (Cavia porcellus).

The authors present eight cases of gastric dilatation and volvulus (GDV) in guinea pigs from the Department and Clinic of Exotic Animal and Wildlife Medicine, University of Veterinary Medicine, Budapest, Hungary between 2012 and 2016. Seven animals were operated on and two survived. Gastric torsion has been noted in many mammalian species. Gastric volvulus has a high morbidity and high mortality rate with a guarded to poor prognosis in all of these species. How GDV develops is still not widely understood. Postmortem examinations, in both our cases and previously reported cases, have failed to reveal the exact causes of the gastric torsions. The aetiology of gastric torsion in guinea pigs is probably multifactorial. Feeding fewer meals per day, eating rapidly, decreased food particle size, exercise, stress after a meal, competition, age, and an aggressive or fearful temperament, are all likely and potential risk factors for GDV development in a similar fashion to dogs. Sex, breeding, dental diseases, anatomical abnormalities, pain and pregnancy may also be contributing factors.

Transiency of retinal ganglion cell action potential responses determined by PSTH time constant.

Retinal ganglion cells (RGC) have been described to react to light stimuli either by producing short bursts of spikes or by maintaining a longer, continuous train of action potentials. Fast, quickly decaying responses are considered to be transient in nature and encode information about movement and direction, while cell responses that show a slow, drawn-out response fall into the sustained category and are thought to be responsible for carrying information related to color and contrast. Multiple approaches have been introduced thus far to measure and determine response transiency. In this study, we adopted and slightly modified a method described by Zeck and Masland to characterize RGC response transiency values and compare them to those obtained by alternative methods. As the first step, RGC spike responses were elicited by light stimulation and peristimulus time histograms (PSTHs) were generated. PSTHs then were used to calculate the time constant (PSTHτ approach). We show that this method is comparable to or more reliable than alternative approaches to describe the temporal characteristics of RGC light responses. In addition, we also show that PSTHτ-s are compatible with time constants measured on RGC and/or bipolar cell graded potentials; thus they are suitable for studying signaling through parallel retinal pathways.

Connexin36 Expression in the Mammalian Retina: A Multiple-Species Comparison.

Much knowledge about interconnection of human retinal neurons is inferred from results on animal models. Likewise, there is a lack of information on human retinal electrical synapses/gap junctions (GJ). Connexin36 (Cx36) forms GJs in both the inner and outer plexiform layers (IPL and OPL) in most species including humans. However, a comparison of Cx36 GJ distribution in retinas of humans and popular animal models has not been presented. To this end a multiple-species comparison was performed in retinas of 12 mammals including humans to survey the Cx36 distribution. Areas of retinal specializations were avoided (e.g., fovea, visual streak, area centralis), thus observed Cx36 distribution differences were not attributed to these species-specific architecture of central retinal areas. Cx36 was expressed in both synaptic layers in all examined retinas. Cx36 plaques displayed an inhomogenous IPL distribution favoring the ON sublamina, however, this feature was more pronounced in the human, swine and guinea pig while it was less obvious in the rabbit, squirrel monkey, and ferret retinas. In contrast to the relative conservative Cx36 distribution in the IPL, the labels in the OPL varied considerably among mammals. In general, OPL plaques were rare and rather small in rod dominant carnivores and rodents, whereas the human and the cone rich guinea pig retinas displayed robust Cx36 labels. This survey presented that the human retina displayed two characteristic features, a pronounced ON dominance of Cx36 plaques in the IPL and prevalent Cx36 plaque conglomerates in the OPL. While many species showed either of these features, only the guinea pig retina shared both. The observed similarities and subtle differences in Cx36 plaque distribution across mammals do not correspond to evolutionary distances but may reflect accomodation to lifestyles of examined species.

Effect of nucleus accumbens lesions on socially motivated behaviour of young domestic chicks.

Behaviour of young domestic chicks when isolated from conspecifics is influenced by two conflicting drives: fear of potential predator and craving for company. The nucleus accumbens (Ac) has been suggested to influence social behaviour, as well as motivation in goal-directed tasks. In this study, the Ac of 1-day-old domestic chicks was lesioned bilaterally, using radiofrequency method. Open field behaviour before and after presenting a silhouette of a bird of prey was recorded, followed by a behavioural test to measure group size preference and social motivation of chicks. Ac-lesioned individuals emitted more distress calls and ambulated more in the open field test, however, they reacted to the predatory stimulus very similarly to control chicks: their vocalization was reduced and the intergroup difference in motor activity also disappeared. There was no difference between the lesioned and control chicks in the latency to approach their conspecifics in the social motivation test, and both groups chose the larger flock (eight) of conspecifics over the smaller one (three). Concerning the role of Ac in social behaviour, a difference between lesioned and sham birds was evident here only in the absence of detectable stimulus (predator or conspecifics). These findings may reflect either decreased fear of exposure to predators or increased craving for conspecifics suggesting that the likely function of Ac is to modulate goal-driven, including socially driven, behaviours, especially when the direct stimulus representing the goal is absent. This is in harmony with the known promotion of impulsiveness by Ac lesions.

Applying games technology to veterinary teaching.

Veterinarian Márton Balogh has been interested in computers, gaming and programming since he was eight years old. Here, he explains how he is using software that is more often associated with the creation of 3D computer games to develop an immersive, virtual consultation room, complete with patient, to help train veterinary students in clinical examination techniques.