Novel Cardiac Mapping Approaches and Multimodal Techniques to Unravel Multidomain Dynamics of Complex Arrhythmias Towards a Framework for Translational Mechanistic-Based Therapeutic Strategies

[ES] Las arritmias cardíacas son un problema importante para los sistemas de salud en el mundo desarrollado debido a su alta incidencia y prevalencia a medida que la población envejece. La fibrilación auricular (FA) y la fibrilación ventricular (FV) se encuentran entre las arritmias más complejas observadas en la práctica clínica. Las consecuencias clínicas de tales alteraciones arrítmicas incluyen el desarrollo de eventos cardioembólicos complejos en la FA, y repercusiones dramáticas debido a procesos fibrilatorios sostenidos que amenazan la vida infringiendo daño neurológico tras paro cardíaco por FV, y que pueden provocar la muerte súbita cardíaca (MSC). Sin embargo, a pesar de los avances tecnológicos de las últimas décadas, sus mecanismos intrínsecos se comprenden de forma incompleta y, hasta la fecha, las estrategias terapéuticas carecen de una base mecanicista suficiente y poseen bajas tasas de éxito. Entre los mecanismos implicados en la inducción y perpetuación de arritmias cardíacas, como la FA, se cree que las dinámicas de las fuentes focales y reentrantes de alta frecuencia, en sus diferentes modalidades, son las fuentes primarias que mantienen la arritmia. Sin embargo, se sabe poco sobre los atractores, así como, de la dinámica espacio-temporal de tales fuentes fibrilatorias primarias, específicamente, las fuentes focales o rotacionales dominantes que mantienen la arritmia. Por ello, se ha desarrollado una plataforma computacional, para comprender los factores (activos, pasivos y estructurales) determinantes, y moduladores de dicha dinámica. Esto ha permitido establecer un marco para comprender la compleja dinámica de los rotores con énfasis en sus propiedades deterministas para desarrollar herramientas basadas en los mecanismos para ayuda diagnóstica y terapéutica. Comprender los procesos fibrilatorios es clave para desarrollar marcadores y herramientas fisiológica- y clínicamente relevantes para la ayuda de diagnóstico temprano. Específicamente, las propiedades espectrales y de tiempo-frecuencia de los procesos fibrilatorios han demostrado resaltar el comportamiento determinista principal de los mecanismos intrínsecos subyacentes a las arritmias y el impacto de tales eventos arrítmicos. Esto es especialmente relevante para determinar el pronóstico temprano de los supervivientes comatosos después de un paro cardíaco debido a fibrilación ventricular (FV). Las técnicas de mapeo electrofisiológico, el mapeo eléctrico y óptico cardíaco, han demostrado ser recursos muy valiosos para dar forma a nuevas hipótesis y desarrollar nuevos enfoques mecanicistas y estrategias terapéuticas mejoradas. Esta tecnología permite además el trabajo multidisciplinar entre clínicos y bioingenieros, para el desarrollo y validación de dispositivos y metodologías para identificar biomarcadores multi-dominio que permitan rastrear con precisión la dinámica de las arritmias identificando fuentes dominantes y atractores con alta precisión para ser dianas de estrategias terapeúticas innovadoras. Es por ello que uno de los objetivos fundamentales ha sido la implantación y validación de nuevos sistemas de mapeo en distintas configuraciones que sirvan de plataforma de desarrollo de nuevas estrategias terapeúticas. Aunque el mapeo panorámico es el método principal y más completo para rastrear simultáneamente biomarcadores electrofisiológicos, su adopción por la comunidad científica es limitada principalmente debido al coste elevado de la tecnología. Aprovechando los avances tecnológicos recientes, nos hemos enfocado en desarrollar, y validar, sistemas de mapeo óptico de alta resolución para registro panorámico cardíaco, utilizando modelos clínicamente relevantes para la investigación básica y la bioingeniería.[CA] Les arítmies cardíaques són un problema important per als sistemes de salut del món desenvolupat a causa de la seva alta incidència i prevalença a mesura que la població envelleix. La fibril·lació auricular (FA) i la fibril·lació ventricular (FV), es troben entre les arítmies més complexes observades a la pràctica clínica. Les conseqüències clíniques d'aquests trastorns arítmics inclouen el desenvolupament d'esdeveniments cardioembòlics complexos en FA i repercussions dramàtiques a causa de processos fibril·latoris sostinguts que posen en perill la vida amb danys neurològics posteriors a la FV, que condueixen a una aturada cardíaca i a la mort cardíaca sobtada (SCD). Tanmateix, malgrat els avanços tecnològics de les darreres dècades, els seus mecanismes intrínsecs s'entenen de forma incompleta i, fins a la data, les estratègies terapèutiques no tenen una base mecanicista suficient i tenen baixes taxes d'èxit. La majoria dels avenços en el desenvolupament de biomarcadors òptims i noves estratègies terapèutiques en aquest camp provenen de tècniques valuoses en la investigació de mecanismes d'arítmia. Entre els mecanismes implicats en la inducció i perpetuació de les arítmies cardíaques, es creu que les fonts primàries subjacents a l'arítmia són les fonts focals reingressants d'alta freqüència dinàmica i AF, en les seves diferents modalitats. Tot i això, se sap poc sobre els atractors i la dinàmica espaciotemporal d'aquestes fonts primàries fibril·ladores, específicament les fonts rotacionals o focals dominants que mantenen l'arítmia. Per tant, s'ha desenvolupat una plataforma computacional per entendre determinants actius, passius, estructurals i moduladors d'aquestes dinàmiques. Això va permetre establir un marc per entendre la complexa dinàmica multidomini dels rotors amb ènfasi en les seves propietats deterministes per desenvolupar enfocaments mecanicistes per a l'ajuda i la teràpia diagnòstiques. La comprensió dels processos fibril·latoris és clau per desenvolupar puntuacions i eines rellevants fisiològicament i clínicament per ajudar al diagnòstic precoç. Concretament, les propietats espectrals i de temps-freqüència dels processos fibril·latoris han demostrat destacar un comportament determinista important dels mecanismes intrínsecs subjacents a les arítmies i l'impacte d'aquests esdeveniments arítmics. Mitjançant coneixements previs, processament de senyals, tècniques d'aprenentatge automàtic i anàlisi de dades, es va desenvolupar una puntuació de risc mecanicista a la aturada cardíaca per FV. Les tècniques de cartografia òptica cardíaca i electrofisiològica han demostrat ser recursos inestimables per donar forma a noves hipòtesis i desenvolupar nous enfocaments mecanicistes i estratègies terapèutiques. Aquesta tecnologia ha permès durant molts anys provar noves estratègies terapèutiques farmacològiques o ablatives i desenvolupar mètodes multidominis per fer un seguiment precís de la dinàmica d'arrímies que identifica fonts i atractors dominants. Tot i que el mapatge panoràmic és el mètode principal per al seguiment simultani de paràmetres electrofisiològics, la seva adopció per part de la comunitat multidisciplinària d'investigació cardiovascular està limitada principalment pel cost de la tecnologia. Aprofitant els avenços tecnològics recents, ens centrem en el desenvolupament i la validació de sistemes de mapes òptics de baix cost per a imatges panoràmiques mitjançant models clínicament rellevants per a la investigació bàsica i la bioenginyeria.[EN] Cardiac arrhythmias are a major problem for health systems in the developed world due to their high incidence and prevalence as the population ages. Atrial fibrillation (AF) and ventricular fibrillation (VF), are amongst the most complex arrhythmias seen in the clinical practice. Clinical consequences of such arrhythmic disturbances include developing complex cardio-embolic events in AF, and dramatic repercussions due to sustained life-threatening fibrillatory processes with subsequent neurological damage under VF, leading to cardiac arrest and sudden cardiac death (SCD). However, despite the technological advances in the last decades, their intrinsic mechanisms are incompletely understood, and, to date, therapeutic strategies lack of sufficient mechanistic basis and have low success rates. Most of the progress for developing optimal biomarkers and novel therapeutic strategies in this field has come from valuable techniques in the research of arrhythmia mechanisms. Amongst the mechanisms involved in the induction and perpetuation of cardiac arrhythmias such AF, dynamic high-frequency re-entrant and focal sources, in its different modalities, are thought to be the primary sources underlying the arrhythmia. However, little is known about the attractors and spatiotemporal dynamics of such fibrillatory primary sources, specifically dominant rotational or focal sources maintaining the arrhythmia. Therefore, a computational platform for understanding active, passive and structural determinants, and modulators of such dynamics was developed. This allowed stablishing a framework for understanding the complex multidomain dynamics of rotors with enphasis in their deterministic properties to develop mechanistic approaches for diagnostic aid and therapy. Understanding fibrillatory processes is key to develop physiologically and clinically relevant scores and tools for early diagnostic aid. Specifically, spectral and time-frequency properties of fibrillatory processes have shown to highlight major deterministic behaviour of intrinsic mechanisms underlying the arrhythmias and the impact of such arrhythmic events. Using prior knowledge, signal processing, machine learning techniques and data analytics, we aimed at developing a reliable mechanistic risk-score for comatose survivors of cardiac arrest due to VF. Cardiac optical mapping and electrophysiological mapping techniques have shown to be unvaluable resources to shape new hypotheses and develop novel mechanistic approaches and therapeutic strategies. This technology has allowed for many years testing new pharmacological or ablative therapeutic strategies, and developing multidomain methods to accurately track arrhymia dynamics identigying dominant sources and attractors. Even though, panoramic mapping is the primary method for simultaneously tracking electrophysiological parameters, its adoption by the multidisciplinary cardiovascular research community is limited mainly due to the cost of the technology. Taking advantage of recent technological advances, we focus on developing and validating low-cost optical mapping systems for panoramic imaging using clinically relevant models for basic research and bioengineering.Calvo Saiz, CJ. (2022). Novel Cardiac Mapping Approaches and Multimodal Techniques to Unravel Multidomain Dynamics of Complex Arrhythmias Towards a Framework for Translational Mechanistic-Based Therapeutic Strategies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182329TESI

Multi-Contrast Photoacoustic Computed Tomography

Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. A stand-alone single-impulse photoacoustic computed tomography (PACT) system has been built, which successfully mitigates these limitations by integrating high spatiotemporal resolution, deep penetration, and full-view fidelity, as well as anatomical, dynamical, and functional contrasts. Based on hemoglobin absorption contrast, the whole-body dynamics and large scale brain functions of rodents have been imaged in real time. The absorption contrast between cytochrome and lipid has enabled PACT to resolve MRI-like whole brain structures. Taking advantage of the distinct absorption signature of melanin, unlabeled circulating melanoma cells have been tracked in real time in vivo. Assisted by near-infrared dyes, the perfusion processes have been visualized in rodents. By localizing single-dyed droplets, the spatial resolution of PACT has been improved by six-fold in vivo. The migration of metallic-based microrobots toward the targeted regions in the intestines has been monitored in real time. Genetically encoded photochromic proteins benefit PACT in detection sensitivity and specificity. The unique photoswitching characteristics of different photochromic proteins allow quantitative multi-contrast imaging at depths. A split version of the photochromic protein has permitted PA detection of protein-protein interactions in deep-seated tumors. The photochromic behaviors have also been utilized to guide photons to form an optical focus inside live tissue. As a rapidly evolving imaging technique, PACT promises pre-clinical applications and clinical translation.</p

High Resolution Multi-parametric Diagnostics and Therapy of Atrial Fibrillation: Chasing Arrhythmia Vulnerabilities in the Spatial Domain

After a century of research, atrial fibrillation (AF) remains a challenging disease to study and exceptionally resilient to treatment. Unfortunately, AF is becoming a massive burden on the health care system with an increasing population of susceptible elderly patients and expensive unreliable treatment options. Pharmacological therapies continue to be disappointingly ineffective or are hampered by side effects due to the ubiquitous nature of ion channel targets throughout the body. Ablative therapy for atrial tachyarrhythmias is growing in acceptance. However, ablation procedures can be complex, leading to varying levels of recurrence, and have a number of serious risks. The high recurrence rate could be due to the difficulty of accurately predicting where to draw the ablation lines in order to target the pathophysiology that initiates and maintains the arrhythmia or an inability to distinguish sub-populations of patients who would respond well to such treatments. There are electrical cardioversion options but there is not a practical implanted deployment of this strategy. Under the current bioelectric therapy paradigm there is a trade-off between efficacy and the pain and risk of myocardial damage, all of which are positively correlated with shock strength. Contrary to ventricular fibrillation, pain becomes a significant concern for electrical defibrillation of AF due to the fact that a patient is conscious when experiencing the arrhythmia. Limiting the risk of myocardial injury is key for both forms of fibrillation. In this project we aim to address the limitations of current electrotherapy by diverging from traditional single shock protocols. We seek to further clarify the dynamics of arrhythmia drivers in space and to target therapy in both the temporal and spatial domain; ultimately culminating in the design of physiologically guided applied energy protocols. In an effort to provide further characterization of the organization of AF, we used transillumination optical mapping to evaluate the presence of three-dimensional electrical substrate variations within the transmural wall during acutely induced episodes of AF. The results of this study suggest that transmural propagation may play a role in AF maintenance mechanisms, with a demonstrated range of discordance between the epicardial and endocardial dynamic propagation patterns. After confirming the presence of epi-endo dyssynchrony in multiple animal models, we further investigated the anatomical structure to look for regional trends in transmural fiber orientation that could help explain the spectrum of observed patterns. Simultaneously, we designed and optimized a multi-stage, multi-path defibrillation paradigm that can be tailored to individual AF frequency content in the spatial and temporal domain. These studies continue to drive down the defibrillation threshold of electrotherapies in an attempt to achieve a pain-free AF defibrillation solution. Finally, we designed and characterized a novel platform of stretchable electronics that provide instrumented membranes across the epicardial surface or implanted within the transmural wall to provide physiological feedback during electrotherapy beyond just the electrical state of the tissue. By combining a spatial analysis of the arrhythmia drivers, the energy delivered and the resulting damage, we hope to enhance the biophysical understanding of AF electrical cardioversion and xiii design an ideal targeted energy delivery protocol to improve upon all limitations of current electrotherapy

Retinal drug delivery: rethinking outcomes for the efficient replication of retinal behavior

The retina is a highly organized structure that is considered to be "an approachable part of the brain." It is attracting the interest of development scientists, as it provides a model neurovascular system. Over the last few years, we have been witnessing significant development in the knowledge of the mechanisms that induce the shape of the retinal vascular system, as well as knowledge of disease processes that lead to retina degeneration. Knowledge and understanding of how our vision works are crucial to creating a hardware-adaptive computational model that can replicate retinal behavior. The neuronal system is nonlinear and very intricate. It is thus instrumental to have a clear view of the neurophysiological and neuroanatomic processes and to take into account the underlying principles that govern the process of hardware transformation to produce an appropriate model that can be mapped to a physical device. The mechanistic and integrated computational models have enormous potential toward helping to understand disease mechanisms and to explain the associations identified in large model-free data sets. The approach used is modulated and based on different models of drug administration, including the geometry of the eye. This work aimed to review the recently used mathematical models to map a directed retinal network.The authors acknowledge the financial support received from the Portuguese Science and Technology Foundation (FCT/MCT) and the European Funds (PRODER/COMPETE) for the project UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020. The authors also acknowledge FAPESP – São Paulo Research Foundation, for the financial support for the publication of the article.info:eu-repo/semantics/publishedVersio

Engineered extracellular vesicles for biomedical applications

Nature's very own nanoparticle, Extracellular vesicles (EVs), are lipid membrane-enclosed vesicles encapsulated with diverse biomolecules and are actively secreted by all cell types for intercellular communication. The unique properties of EVs, such as stability in circulation, biocompatibility, immune tolerance, and the ability to cross biological barriers, render EVs a next-generation drug delivery tool. Therapeutic EV research has seen tremendous development in the past decade, from in vitro studies towards pre-clinical models to various clinical trials. Even so, the road towards successful clinical translation has faced various hurdles primarily due to the lack of technology to address the knowledge gap in EV biology. Hence, this thesis is focused on addressing some of these critical challenges and exploring novel biomedical applications for EVs. EVs are considered as essential mediators in physiology and disease pathology. However, to elucidate their important role in pathophysiology or as therapeutics, sensitive tools for visualising them are much needed. Here, in paper I, we have developed a sensitive bioluminescent labelling system for tracking EVs in vitro and in vivo. By genetically modifying the producer cells with EV-associated tetraspanins-fusions, we could efficiently load luciferase enzymes (Nanoluciferase and Thermoluciferase) into EVs. Utilising the Nanoluciferase labelling system, we could detect as low as 5000 EVs in a solution, and the naked eye could visualise the luminescence generated from these EVs. With this level of sensitivity, we explored various in vivo applications and observed that exogenous EVs are rapidly distributed throughout the body, primarily to the liver, lung, and spleen. In addition, we identified that EV subpopulations differ in their in vivo biodistribution profile. In summary, this system allows for highly sensitive detection of EVs in vivo and reflects the true fate of EVs. Despite tremendous advancement in understanding EV biology or engineering, techniques to surface engineer EVs with large protein biotherapeutics without altering their innate properties are largely lacking. Here in paper II, we developed a novel surface display technology for EVs, which allows for efficient display of several membrane proteins on the EV surface simultaneously. Using this platform, we decorated EVs' surface with cytokine receptors that can decoy pro-inflammatory cytokines such as TNF-α or IL-6/sIL-6R complexes. These cytokine decoy EVs were more active than a clinically approved biologic against TNF-α in vitro. Importantly, these cytokine decoy EVs ameliorated the disease phenotype in three different mice inflammation models, including neuroinflammation. In paper III, we have applied interleukin 6 signal transducer (IL-6ST) decoy EVs to tackle inflammation in muscle pathologies to enhance the muscle regeneration process. Using decoy EVs as a therapeutic intervention in mdx mice mimicking Duchene Muscular Dystrophy (DMD), we could achieve significant downregulation of phosphorylation of the proinflammatory transcription factor STAT3 in muscles. In conclusion, the tools developed in this thesis, from highly sensitive detection of EV subtype to efficient display of biotherapeutics cargo on EV surfaces, holds great future potential and applicability in numerous biomedical applications of EVs

Cellular, molecular and synaptic properties of pain: A microfluidic approach

The detection of stimuli that produce painful behaviors occurs within a subset of sensory neurons, namely nociceptors. The signals elicited by nociceptors are transmitted to the spinal cord through synaptic relay points in the dorsal horn. Nociceptors undergo significant plastic changes upon nerve injury and inflammation, where they augment painful signaling and can further modulate the excitability of central neurons, resulting in persistent pain. Elucidating the cellular and molecular properties of the somatosensory apparatus provides a deeper understanding of the physiology of painful signaling and opens therapeutic contingencies for the treatment of painful conditions. However, the plasticity of nociceptors and their contribution to painful pathologies has not been fully captured by existing methodologies. The current in vivo behavioral and electrophysiological techniques lack the spatial resolution to detect molecular contributions to nociceptor excitability, while in vitro studies have focused on the cell bodies of nociceptors and not the axons, that often represent the actual sites of injury.To address this problem, we have employed a novel cell culture model which exploits advances in microfluidics. Microfluidic cultures allow the investigation of the functional properties of nociceptor axons separately from their respective cell bodies. Three problems are addressed using variations of this technique in my thesis. First, the properties that the inflammatory mediator prostaglandin E2 (PGE2) exerts on sensory axons are elucidated. Second, the periphery-to-center circuit between DRG and DH neurons is recapitulated and the formation of functional synapses between them is reported. Third, the pre-synaptic properties of transient receptor potential (TRP) and voltage-gated sodium (Nav) channels are examined and subsequently their contribution to DH excitability between axotomized and non-axotomized cultures is compared.The results from the first investigation reveal a direct and persistent depolarization of sensory axons by PGE2. It is shown that the PGE2-evoked activity is dependent on the EP4/cAMP/PKA pathway. Evidence is provided for the mediation of this activity by the TTX-resistant sodium channel Nav1.8 and by the calcium activated chloride channel ANO1. Hence, it is proposed that PGE2 possesses a dual role as a sensitizer, but also as direct activator of sensory axons. The molecular mechanisms that underpin the latter phenomenon are delineated.In the second investigation, mouse postnatal DRG and DH neurons are co-cultured in microfluidic devices. The co-culture model recapitulates salient features of synaptic transmission between DRG and DH, allowing the recording of DH neuron activity following DRG neuron stimulation. The model is important because it allows a spatially resolved interrogation of the molecular properties of nociceptors and the extent to which they contribute to synaptic transmission.Last, the co-culture model is employed to investigate the contribution of pre-synaptic TRP and Nav channels to synaptic transmission. As a surrogate model of nerve transection, an axotomy is introduced and compared with the contribution of these channels under intact conditions. A role for Nav1.7 channels in synaptic transmission under intact conditions is reported, however it is lost following axotomy. Surprisingly, Nav1.6 channels emerge as principal contributors to synaptic transmission only following axotomy. A novel finding of this thesis regards the pre-synaptic role of TRPV1 and TRPA1 channels. Traditionally considered as transducer channels of thermal and chemical noxious stimuli, these channels show significant pre-synaptic activity where they dynamically contribute to synaptic transmission, a property which is lost following axotomy.Overall, this thesis demonstrates the versatility of the microfluidic platform and unravels novel properties of a neuroimmune interaction relevant to inflammatory pain, synaptic transmission between DRG and DH neurons and finally, the pre-synaptic contribution of TRP and Nav channels in normal and axotomized conditions. The delineation of the molecular properties of nociceptors using pharmacological or genetic manipulations at distinct anatomical parts may further reveal unknown aspects about the physiology and pathophysiology of these neurons. The platform possesses an industrial utility too, as it can be used for the screening of novel analgesic molecules and the determination of how they affect the excitability of nociceptors or DH neurons

Engineering, applications, and future perspectives of GPCR-based genetically encoded fluorescent indicators for neuromodulators

This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity

Architecture of the Mouse Brain Synaptome

Synapses are found in vast numbers in the brain and contain complex proteomes. We developed genetic labeling and imaging methods to examine synaptic proteins in individual excitatory synapses across all regions of the mouse brain. Synapse catalogs were generated from the molecular and morphological features of a billion synapses. Each synapse subtype showed a unique anatomical distribution, and each brain region showed a distinct signature of synapse subtypes. Whole-brain synaptome cartography revealed spatial architecture from dendritic to global systems levels and previously unknown anatomical features. Synaptome mapping of circuits showed correspondence between synapse diversity and structural and functional connectomes. Behaviorally relevant patterns of neuronal activity trigger spatiotemporal postsynaptic responses sensitive to the structure of synaptome maps. Areas controlling higher cognitive function contain the greatest synapse diversity, and mutations causing cognitive disorders reorganized synaptome maps. Synaptome technology and resources have wide-ranging application in studies of the normal and diseased brain

Extracellular electrophysiology with close-packed recording sites: spike sorting and characterization

Advances in recording technologies now allow us to record populations of neurons simultaneously, data necessary to understand the network dynamics of the brain. Extracellular probes are fabricated with ever greater numbers of recording sites to capture the activity of increasing numbers of neurons. However, the utility of this extracellular data is limited by an initial analysis step, spike sorting, that extracts the activity patterns of individual neurons from the extracellular traces. Commonly used spike sorting methods require manual processing that limits their scalability, and errors can bias downstream analyses. Leveraging the replication of the activity from a single neuron on nearby recording sites, we designed a spike sorting method consisting of three primary steps: (1) a blind source separation algorithm to estimate the underlying source components, (2) a spike detection algorithm to find the set of spikes from each component best separated from background activity and (3) a classifier to evaluate if a set of spikes came from one individual neuron. To assess the accuracy of our method, we simulated multi-electrode array data that encompass many of the realistic variations and the sources of noise in in vivo neural data. Our method was able to extract individual simulated neurons in an automated fashion without any errors in spike assignment. Further, the number of neurons extracted increased as we increased recording site count and density. To evaluate our method in vivo, we performed both extracellular recording with our close-packed probes and a co-localized patch clamp recording, directly measuring one neuron’s ground truth set of spikes. Using this in vivo data we found that when our spike sorting method extracted the patched neuron, the spike assignment error rates were at the low end of reported error rates, and that our errors were frequently the result of failed spike detection during bursts where spike amplitude decreased into the noise. We used our in vivo data to characterize the extracellular recordings of burst activity and more generally what an extracellular electrode records. With this knowledge, we updated our spike detector to capture more burst spikes and improved our classifier based on our characterizations