Cardiomyocyte electrophysiology and its modulation: current views and future prospects.

Normal and abnormal cardiac rhythms are of key physiological and clinical interest. This introductory article begins from Sylvio Weidmann's key historic 1950s microelectrode measurements of cardiac electrophysiological activity and Singh & Vaughan Williams's classification of cardiotropic targets. It then proceeds to introduce the insights into cardiomyocyte function and its regulation that subsequently emerged and their therapeutic implications. We recapitulate the resulting view that surface membrane electrophysiological events underlying cardiac excitation and its initiation, conduction and recovery constitute the final common path for the cellular mechanisms that impinge upon this normal or abnormal cardiac electrophysiological activity. We then consider progress in the more recently characterized successive regulatory hierarchies involving Ca2+ homeostasis, excitation-contraction coupling and autonomic G-protein signalling and their often reciprocal interactions with the surface membrane events, and their circadian rhythms. Then follow accounts of longer-term upstream modulation processes involving altered channel expression, cardiomyocyte energetics and hypertrophic and fibrotic cardiac remodelling. Consideration of these developments introduces each of the articles in this Phil. Trans. B theme issue. The findings contained in these articles translate naturally into recent classifications of cardiac electrophysiological targets and drug actions, thereby encouraging future iterations of experimental cardiac electrophysiological discovery, and testing directed towards clinical management. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Investigating Neural Correlates of Behavior Through In Vivo Electrophysiology.

Behavioral neuroscience has long relied on in vivo electrophysiology to provide spatially and temporally precise answers to complex questions about the neural dynamics underlying sensory processing and action execution. Investigating the neural correlates of behavior can be challenging in freely behaving animals, especially when making inferences related to internal states that are temporally or conceptually ambiguous, such as decision-making or motivation. This necessitates careful creation of appropriate and rigorous controls and awareness of the many potential confounds when attributing neural signals to animal behavior. This article discusses fundamental considerations for the optimal design and interpretation of in vivo rodent electrophysiological recording experiments and focuses on the different optimization strategies required when investigating neural encoding of external stimuli versus free behavior. The first protocol offers suggestions specific to intracranial surgical implantation of multielectrode arrays. The second protocol delves into optimization strategies and tips useful for designing and interpreting recording experiments conducted in freely behaving rodents. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Surgical implantation of the multielectrode array Basic Protocol 2: Optimizing experimental design and parameters.

Large-scale electrophysiology and deep learning reveal distorted neural signal dynamics after hearing loss.

Listeners with hearing loss often struggle to understand speech in noise, even with a hearing aid. To better understand the auditory processing deficits that underlie this problem, we made large-scale brain recordings from gerbils, a common animal model for human hearing, while presenting a large database of speech and noise sounds. We first used manifold learning to identify the neural subspace in which speech is encoded and found that it is low-dimensional and that the dynamics within it are profoundly distorted by hearing loss. We then trained a deep neural network (DNN) to replicate the neural coding of speech with and without hearing loss and analyzed the underlying network dynamics. We found that hearing loss primarily impacts spectral processing, creating nonlinear distortions in cross-frequency interactions that result in a hypersensitivity to background noise that persists even after amplification with a hearing aid. Our results identify a new focus for efforts to design improved hearing aids and demonstrate the power of DNNs as a tool for the study of central brain structures.

Learnable latent embeddings for joint behavioural and neural analysis.

Mapping behavioural actions to neural activity is a fundamental goal of neuroscience. As our ability to record large neural and behavioural data increases, there is growing interest in modelling neural dynamics during adaptive behaviours to probe neural representations1-3. In particular, although neural latent embeddings can reveal underlying correlates of behaviour, we lack nonlinear techniques that can explicitly and flexibly leverage joint behaviour and neural data to uncover neural dynamics3-5. Here, we fill this gap with a new encoding method, CEBRA, that jointly uses behavioural and neural data in a (supervised) hypothesis- or (self-supervised) discovery-driven manner to produce both consistent and high-performance latent spaces. We show that consistency can be used as a metric for uncovering meaningful differences, and the inferred latents can be used for decoding. We validate its accuracy and demonstrate our tool's utility for both calcium and electrophysiology datasets, across sensory and motor tasks and in simple or complex behaviours across species. It allows leverage of single- and multi-session datasets for hypothesis testing or can be used label free. Lastly, we show that CEBRA can be used for the mapping of space, uncovering complex kinematic features, for the production of consistent latent spaces across two-photon and Neuropixels data, and can provide rapid, high-accuracy decoding of natural videos from visual cortex.

[INTRAOPERATIVE ELECTROPHYSIOLOGY MAPPING OF CORTICAL FUNCTION: A BRIEF HISTORY AND EVOLVING ALTERNATIVE].

INTRODUCTION: Multiple studies have demonstrated that the improved extent of resection for patients with glioma is associated with improved survival. The use of intraoperative electrophysiology cortical mapping to demonstrate function became a standard of care in modern neurosurgery and an indispensable tool to achieve the goal of maximal safe resection in tumor surgery. In this study, we review the brief history of intraoperative electrophysiology cortical mapping from the first cortical mapping study back in 1870 to the innovative tool of broad gamma cortical mapping used today.

DNA methylome and transcriptome profiling reveal key electrophysiology and immune dysregulation in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM.

Association between circulating leukocytes and arrhythmias: Mendelian randomization analysis in immuno-cardiac electrophysiology.

Background: Cardiac arrhythmia is a common disease associated with high mortality and morbidity. Circulating leukocyte counts, which serve as a biomarker for assessing systemic immune status, have been linked to arrhythmias in observational studies. However, observational studies are plagued by confounding factors and reverse causality, whether alterations in circulating leukocyte components are causally associated with arrhythmias remains uncertain. The present study explored this question based on genetic evidence. Methods and findings: We performed Mendelian randomization (MR) analysis to evaluate whether alterations in leukocyte counts affect aggregated risk of all types of arrhythmia or risk of five specific types of arrhythmia. Single-nucleotide polymorphisms serving as proxies for leukocyte differential counts were retrieved from the Blood Cell Consortium, and statistical data on arrhythmias were obtained from the UK Biobank), FinnGenand a meta-analysis of genome-wide association studies for atrial fibrillation. We applied inverse variance-weighted method as the primary analysis, complemented by a series of sensitivity analyses. Bidirectional analyses were conducted to assess reverse causality. Finally, multivariable MR was performed to study the joint effects of multiple risk factors. We found that genetically predicted differential leukocyte counts were not significantly associated with aggregated occurrence of all types of arrhythmia. In contrast, each 1-standard deviation increase in lymphocyte count was associated with 46% higher risk of atrioventricular block (OR 1.46, 95% CI 1.11-1.93, p=0.0065). A similar effect size was observed across all MR sensitivity analyses, with no evidence of horizontal pleiotropy. Reverse MR analysis suggested that atrioventricular block was unlikely to cause changes in lymphocyte count. Primary MR analysis based on the inverse-variance weighted method suggested that changes in neutrophil count alter risk of right bundle branch block, and changes in basophil count alter risk of atrial fibrillation. However, these causal relationships were not robust in sensitivity analyses. We found no compelling evidence that neutrophil or lymphocyte counts cause atrial fibrillation. Conclusion: Our data support higher lymphocyte count as a causal risk factor for atrioventricular block. These results highlight the importance of immune cells in the pathogenesis of specific cardiac conduction disorders.

Proposing national diagnostic reference levels for electrophysiology studies and catheter ablation procedures in Bulgaria.

INTRODUCTION: The implementation of diagnostic reference levels (DRLs) is an essential tool for optimisation of the routine practice, better management of patient exposure while maintaining sufficient image quality. National DRLs for electrophysiology (EP) procedures are not available in our country. PURPOSE: The main purpose of the study was to propose, for first time in Bulgaria, national DRLs (NDRLs) for EP studies and ablation procedures of two different levels of complexity. The proposed DRLs can be later used to establish NDRLs by the national authority with regulatory functions related to medical exposure. METHOD: A retrospective study was done with the three highest volume Bulgarian EP centers, where over 95% of all cardiac ablations were performed. Data were extracted from the electronic registry for invasive electrophysiology BG-EPHY. Independently of the proposed NDRLs, we also compared the air kerma-area product (KAP) between the participating centers for procedures of the same level of complexity. RESULTS: The proposed NDRL in terms of KAP were: 5.2 Gy.cm2 for diagnostic EP studies, 25.5 Gy.cm2 for simple ablations, and 52.1 Gy.cm2 for complex ablations. There was a significant variation in KAP for procedures with the same degree of complexity within each center. CONCLUSION: This study is the first to propose NDLRs for EP studies and ablation procedures of two levels of complexity in Bulgaria. The results identified EP procedures requiring further optimization of patient protection and provided a basis for future comparisons and standardization with further investigations on the topic. The proposed NDRLs are recommended to be used for better management of radiation exposure during EP procedures of different levels of complexity.

Sleep fMRI with simultaneous electrophysiology at 9.4 T in male mice.

Sleep is ubiquitous and essential, but its mechanisms remain unclear. Studies in animals and humans have provided insights of sleep at vastly different spatiotemporal scales. However, challenges remain to integrate local and global information of sleep. Therefore, we developed sleep fMRI based on simultaneous electrophysiology at 9.4 T in male mice. Optimized un-anesthetized mouse fMRI setup allowed manifestation of NREM and REM sleep, and a large sleep fMRI dataset was collected and openly accessible. State dependent global patterns were revealed, and state transitions were found to be global, asymmetrical and sequential, which can be predicted up to 17.8 s using LSTM models. Importantly, sleep fMRI with hippocampal recording revealed potentiated sharp-wave ripple triggered global patterns during NREM than awake state, potentially attributable to co-occurrence of spindle events. To conclude, we established mouse sleep fMRI with simultaneous electrophysiology, and demonstrated its capability by revealing global dynamics of state transitions and neural events.

Cardiogenic control of affective behavioural state.

Emotional states influence bodily physiology, as exemplified in the top-down process by which anxiety causes faster beating of the heart1-3. However, whether an increased heart rate might itself induce anxiety or fear responses is unclear3-8. Physiological theories of emotion, proposed over a century ago, have considered that in general, there could be an important and even dominant flow of information from the body to the brain9. Here, to formally test this idea, we developed a noninvasive optogenetic pacemaker for precise, cell-type-specific control of cardiac rhythms of up to 900 beats per minute in freely moving mice, enabled by a wearable micro-LED harness and the systemic viral delivery of a potent pump-like channelrhodopsin. We found that optically evoked tachycardia potently enhanced anxiety-like behaviour, but crucially only in risky contexts, indicating that both central (brain) and peripheral (body) processes may be involved in the development of emotional states. To identify potential mechanisms, we used whole-brain activity screening and electrophysiology to find brain regions that were activated by imposed cardiac rhythms. We identified the posterior insular cortex as a potential mediator of bottom-up cardiac interoceptive processing, and found that optogenetic inhibition of this brain region attenuated the anxiety-like behaviour that was induced by optical cardiac pacing. Together, these findings reveal that cells of both the body and the brain must be considered together to understand the origins of emotional or affective states. More broadly, our results define a generalizable approach for noninvasive, temporally precise functional investigations of joint organism-wide interactions among targeted cells during behaviour.

CFTR function, pathology and pharmacology at single-molecule resolution.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Solid-Supported Membrane (SSM)-Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE²R) in Early Drug Discovery.

This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE²R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE²R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cell-line-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE²R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM ) and maximal velocity (Vmax ) of substrate uptake. © 2023 Sanofi. Current Protocols published by Wiley Periodicals LLC. PROTOCOL GROUP 1: Sample preparation for SSM-based electrophysiology assays Support Protocol 1: Production of cell batches Support Protocol 2: Simple isolation of cell membranes Alternate Protocol 1: Isolation of cell membranes with sucrose gradient pre-purification Support Protocol 3: Production and isolation of liposomes Support Protocol 4: Preparation of sensor with isolated cell membranes Alternate Protocol 2: Preparation of sensor with isolated proteoliposomes PROTOCOL GROUP 2: Determination of assay parameters for SSM-based electrophysiology assay Support Protocol 5: Assay with stable buffer Alternate Protocol 3: Assay with ion gradient Support Protocol 6: Determination of membrane/liposome concentration Support Protocol 7: Determination of substrate dependency kM PROTOCOL GROUP 3: Determination of advanced assay parameters for SSM-based electrophysiology assays Support Protocol 8: Assessment of ion concentration dependency Support Protocol 9: Assessment of pH dependency Support Protocol 10: Assessment of DMSO dependency Support Protocol 11: Assessment of signal stability with multiple activations PROTOCOL GROUP 4: Compound testing through SSM-based electrophysiology assays using SURFE²R apparatus Support Protocol 12: Assessment of signal specificity of a published inhibitor or unknown compound(s) Support Protocol 13: Compound wash-out Support Protocol 14: Statistical analysis.

Stress, depression, and hippocampus: from biochemistry to electrophysiology.

Major depressive disorder is a very common serious mental illness with increasing prevalence in the population. Its pathology includes biochemical, morphological, and electrophysiological changes in various brain areas. In spite of decades of extensive research pathophysiology of depression is still not sufficiently understood. When depression occurs just before or during pregnancy, it may have a detrimental effect on perinatal and/or postnatal brain development, affecting the offspring's behavior. An important role in the pathology of depression is the hippocampus as a center for cognition and memory. Here we review changes in morphology, biochemical, and electrical signaling caused by depression in first and second generation identified in various animal models.

A mesh microelectrode array for non-invasive electrophysiology within neural organoids.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.

BACKGROUND: Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. METHODS: Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. KEY RESULTS: Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. CONCLUSIONS: Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.

Experimental Verification for Numerical Simulation of Thalamic Stimulation-Evoked Calcium-Sensitive Fluorescence and Electrophysiology with Self-Assembled Multifunctional Optrode.

Owing to its capacity to eliminate a long-standing methodological limitation, fiber photometry can assist research gaining novel insight into neural systems. Fiber photometry can reveal artifact-free neural activity under deep brain stimulation (DBS). Although evoking neural potential with DBS is an effective method for mediating neural activity and neural function, the relationship between DBS-evoked neural Ca2+ change and DBS-evoked neural electrophysiology remains unknown. Therefore, in this study, a self-assembled optrode was demonstrated as a DBS stimulator and an optical biosensor capable of concurrently recording Ca2+ fluorescence and electrophysiological signals. Before the in vivo experiment, the volume of tissue activated (VTA) was estimated, and the simulated Ca2+ signals were presented using Monte Carlo (MC) simulation to approach the realistic in vivo environment. When VTA and the simulated Ca2+ signals were combined, the distribution of simulated Ca2+ fluorescence signals matched the VTA region. In addition, the in vivo experiment revealed a correlation between the local field potential (LFP) and the Ca2+ fluorescence signal in the evoked region, revealing the relationship between electrophysiology and the performance of neural Ca2+ concentration behavior. Concurrent with the VTA volume, simulated Ca2+ intensity, and the in vivo experiment, these data suggested that the behavior of neural electrophysiology was consistent with the phenomenon of Ca2+ influx to neurons.

Temporal profiles of neuronal responses to repeated tone stimuli in the mouse primary auditory cortex.

How the auditory system processes temporal information of sound has been investigated extensively using repeated stimuli. Recent studies on how the response of neurons in the primary auditory cortex (A1) changes with the progression of stimulus repetition, have reported response temporal profiles of two categories: "adaptation", i.e., gradual decrease, and "facilitation", i.e., gradual increase. To explore the existence of profiles of other categories and to examine the tone-frequency-dependence of the profile category in single neurons, here we studied the response of mouse A1 neurons to four or five tone-trains; each train comprised 10 identical tone pips, with 0.5-s inter-tone-intervals, and the four or five trains differed only in tone frequency. The response to each tone in a train was evaluated using the peak of the ON response, and how the peak response changed with the tone number in the train, i.e., the response temporal profile, was examined. We confirmed the existence of profiles of both "adaptation" and "facilitation" categories; "adaptation" could be further subcategorized into "slow adaptation" and "fast adaptation" profiles, with the latter being encountered more frequently. Moreover, two new categories of non-monotonic profiles were identified: an "adaptation with recovery" profile and a "facilitation followed by adaptation" profile. Examination of single neurons with trains of different tone frequencies revealed that some A1 neurons exhibited profiles of the same category to tone trains of different tone frequencies, whereas others exhibited profiles of different categories, depending on the tone frequency. These results demonstrate the variety in the response temporal profiles of mouse A1 neurons, which may benefit the encoding of individual tones in a train.