Depolarization Events Research Articles

Neurosurgical Spreading Depolarization Monitoring: Why, How, and What to Do About It.

Monitoring of electrocorticography for the purpose of detecting spreading depolarization (SD) events is becoming increasingly used both for research and clinical applications. Although such monitoring bears many similarities to standard long-term epilepsy monitoring, there are a number of differences that neurosurgeons need to be aware of when initiating such a program. In addition, most of the focus in SD monitoring has been on traumatic and vascular conditions, where invasive monitoring is used commonly, but electrocorticography is not commonly used. In this brief review, a clinically focused approach is presented to support successful monitoring. This is not intended to be a comprehensive or systematic description of the neuroscience of SD and its clinical relevance, as many such previous reviews exist. It is also recognized that this is a rapidly evolving field and that new advances may disrupt these approaches and that there is a diversity of opinion on these topics, even among SD experts. Nonetheless, the general approach to SD monitoring has now been in use for >15 years and is the basis for several active and proposed clinical trials (NCT05337618, NCT04966546), so an understanding from a neurosurgical perspective of the rationale and approach to monitoring is warranted. In this review, we will consider the potential indications for SD monitoring in clinical trials or clinical care, the methodology for recording and interpreting, and finally some potential therapeutic approaches that are being considered in patients with clinically detected SD.

A dangerous liaison: Spreading depolarization and tissue acidification in cerebral ischemia.

Brain pH is precisely regulated, and pH transients associated with activity are rapidly restored under physiological conditions. During ischemia, the brain's ability to buffer pH changes is rapidly depleted. Tissue oxygen deprivation causes a shift from aerobic to anaerobic metabolism and the accumulation of lactic acid and protons. Although the degree of tissue acidosis resulting from ischemia depends on the severity of the ischemia, spreading depolarization (SD) events emerge as central elements to determining ischemic tissue acidosis. A marked decrease in tissue pH during cerebral ischemia may exacerbate neuronal injury, which has become known as acidotoxicity, in analogy to excitotoxicity. The cellular pathways underlying acidotoxicity have recently been described in increasing detail. The molecular structure of acid or base carriers and acidosis-activated ion channels, the precise (dys)homeostatic conditions under which they are activated, and their possible role in severe ischemia have been addressed. The expanded understanding of acidotoxic mechanisms now provides an opportunity to reevaluate the contexts that lead to acidotoxic injury. Here, we review the specific cellular pathways of acidotoxicity and demonstrate that SD plays a central role in activating the molecular machinery leading to acid-induced damage. We propose that SD is a key contributor to acidotoxic injury in cerebral ischemia.

The Impact of Sedative Choice in the Management of Aneurysmal Subarachnoid Hemorrhage: A Scoping Review.

Aneurysmal subarachnoid hemorrhage (aSAH) is characterized by high mortality and morbidity. This scoping review assesses the current evidence regarding the use of sedatives and analgesics in the acute intensive care unit management of aSAH. We conducted a systematic search of Ovid MEDLINE, Ovid Embase, Ovid EmCare, APA PsycInfo, CINAHL, and the Cochrane Database of Systematic Reviews from inception to June 2023. Studies were included if they enrolled intensive care unit patients aged 18 or older with a significant proportion (> 20%) who had aSAH and evaluated the impact of one or more commonly used analgosedatives on physiological parameters in the management of aSAH. The methodological quality of the studies was assessed using the Methodological Index for Nonrandomized Studies score. Of 2,583 articles, 11 met the inclusion criteria. The median sample size was 47 (interquartile range 10-127), and the median Methodological Index for Nonrandomized Studies score was 9.5 (interquartile range 8-11). The studies' publication years ranged from 1980 to 2023. Dexmedetomidine and ketamine showed potential benefits in reducing the incidence of cortical spreading depolarization and delayed cerebral ischemia. Propofol and opioids appeared safe but lacked robust evidence for efficacy. Benzodiazepines were associated with increased delayed cerebral ischemia-related cerebral infarctions and cortical spreading depolarization events. The evidence available to guide the use of analgosedative medications in aSAH is critically inadequate. Dexmedetomidine and ketamine warrant further exploration in large-scale prospective studies because of their potential benefits. Improved study designs with consistent definitions and a focus on patient-centered outcomes are necessary to inform clinical practice.

Oxygen-Based Autoregulation Indices Associated with Clinical Outcomes and Spreading Depolarization in Aneurysmal Subarachnoid Hemorrhage.

Impairment in cerebral autoregulation has been proposed as a potentially targetable factor in patients with aneurysmal subarachnoid hemorrhage (aSAH); however, there are different continuous measures that can be used to calculate the state of autoregulation. In addition, it has previously been proposed that there may be an association of impaired autoregulation with the occurrence of spreading depolarization (SD) events. Study participants with invasive multimodal monitoring and aSAH were enrolled in an observational study. Autoregulation indices were prospectively calculated from this database as a 10s moving correlation coefficient between various cerebral blood flow (CBF) surrogates and mean arterial pressure (MAP). In study participants with subdural electrocorticography (ECoG) monitoring, SD was also scored. Associations between clinical outcomes using the modified Rankin scale and occurrence of either isolated or clustered SD were assessed. A total of 320 study participants were included, 47 of whom also had ECoG SD monitoring. As expected, baseline severity factors, such as modified Fisher scale score and World Federation of Neurosurgical Societies scale grade, were strongly associated with the clinical outcome. SD probability was related to blood pressure in a triphasic pattern, with a linear increase in probability below MAP of ~ 100 mm Hg. Multiple autoregulation indices were available for review based on moving correlations between mean arterial pressure (MAP) and various surrogates of cerebral blood flow (CBF). We calculated the pressure reactivity (PRx) using two different sources for intracranial pressure (ICP). We calculated the oxygen reactivity (ORx) using the partial pressure of brain tissue oxygen (PbtO2) from the Licox probe. We calculated the cerebral blood flow reactivity (CBFRx) using perfusion measurements from the Bowman perfusion probe. Finally, we calculated the cerebral oxygen saturation reactivity (OSRx) using regional cerebral oxygen saturation measured by near-infrared spectroscopy from the INVOS sensors. Only worse ORx and OSRx were associated with worse clinical outcomes. Both ORx and OSRx also were found to increase in the hour prior to SD for both sporadic and clustered SD. Impairment in autoregulation in aSAH is associated with worse clinical outcomes and occurrence of SD when using ORx and OSRx. Impaired autoregulation precedes SD occurrence. Targeting the optimal MAP or cerebral perfusion pressure in patients with aSAH should use ORx and/or OSRx as the input function rather than intracranial pressure.

Tissue-specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart.

Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

Cerebral autoregulation, spreading depolarization, and implications for targeted therapy in brain injury and ischemia.

Cerebral autoregulation is an intrinsic myogenic response of cerebral vasculature that allows for preservation of stable cerebral blood flow levels in response to changing systemic blood pressure. It is effective across a broad range of blood pressure levels through precapillary vasoconstriction and dilation. Autoregulation is difficult todirectly measure and methods to indirectly ascertain cerebral autoregulation status inherently require certain assumptions. Patients with impaired cerebral autoregulation may be at risk of brain ischemia. One of the central mechanisms of ischemia in patients with metabolically compromised states is likely the triggering of spreading depolarization (SD) events and ultimately, terminal (or anoxic) depolarization. Cerebral autoregulation and SD are therefore linked when considering the risk of ischemia. In this scoping review, we will discuss the range of methods to measure cerebral autoregulation, their theoretical strengths and weaknesses, and the available clinical evidence to support their utility. We will then discuss the emerging link between impaired cerebral autoregulation and the occurrence of SD events. Such an approach offers the opportunity to better understand an individual patient's physiology and provide targeted treatments.

Effects of noninvasive neuromodulation targeting the spinal cord on early learning of force control by the digits.

Control of finger forces underlies our capacity for skilled hand movements acquired during development and reacquired after neurological injury. Learning force control by the digits, therefore, predicates our functional independence. Noninvasive neuromodulation targeting synapses that link corticospinal neurons onto the final common pathway via spike-timing-dependent mechanisms can alter distal limb motor output on a transient basis, yet these effects appear subject to individual differences. Here, we investigated how this form of noninvasive neuromodulation interacts with task repetition to influence early learning of force control during precision grip. The unique effects of neuromodulation, task repetition, and neuromodulation coinciding with task repetition were tested in three separate conditions using a within-subject, cross-over design (n = 23). We found that synchronizing depolarization events within milliseconds of stabilizing precision grip accelerated learning but only after accounting for individual differences through inclusion of subjects who showed upregulated corticospinal excitability at 2 of 3 time points following conditioning stimulation (n = 19). Our findings provide insights into how the state of the corticospinal system can be leveraged to drive early motor skill learning, further emphasizing individual differences in the response to noninvasive neuromodulation. We interpret these findings in the context of biological mechanisms underlying the observed effects and implications for emerging therapeutic applications.

Characterization of neuromuscular transmission and projections of muscle motor neurons in the rat stomach.

The stomach is the primary reservoir of the gastrointestinal tract, where ingested content is broken down into small particles. Coordinated relaxation and contraction is essential for rhythmic motility and digestion, but how the muscle motor innervation is organized to provide appropriate graded regional control is not established. In this study, we recorded neuromuscular transmission to the circular muscle using intracellular microelectrodes to investigate the spread of the influence of intrinsic motor neurons. In addition, microanatomical investigations of neuronal projections and pharmacological analysis were conducted to investigate neuromuscular relationships. We found that inhibitory neurotransmission to the circular muscle is graded with stimulus strength and circumferential distance from the stimulation site. The influence of inhibitory neurons declined between 1 and 11 mm from the stimulation site. In the antrum, corpus, and fundus, the declines at 11 mm were about 20%, 30%, and 50%, respectively. Stimulation of inhibitory neurons elicited biphasic hyperpolarizing potentials often followed by prolonged depolarizing events in the distal stomach, but only hyperpolarizing events in the proximal stomach. Excitatory neurotransmission influence varied greatly between proximal stomach, where depolarizing events occurred, and distal stomach, where no direct electrical effects in the muscle were observed. Structural studies using microlesion surgeries confirmed a dominant circumferential projection. We conclude that motor neuron influences extend around the gastric circumference, that the effectiveness can be graded by the recruitment of different numbers of motor neuron nerve terminals to finely control gastric motility, and that the ways in which the neurons influence the muscle differ between anatomical regions.NEW & NOTEWORTHY This study provides a detailed mapping of nerve transmission to the circular muscle of the different anatomical regions of rat stomach. It shows that excitatory and inhibitory influences extend around the gastric circumference and that there is a summation of neural influence that allows for finely graded control of muscle tension and length. Nerve-mediated electrical events are qualitatively and quantitatively different between regions, for example, excitatory neurons have direct effects on fundus but not antral muscle.

Abstract 12942: The D801N Variant in ATP1A3 -Encoded Na/K-atpase Alpha 3 Shortens the Cardiac Myocyte Action Potential and Increases Risk for Calcium Mediated Arrhythmias

Background: Alternating hemiplegia of childhood (AHC) is a rare de novo syndrome that manifests with episodic hemiplegia, seizures, dystonia, and, notably, sudden unexplained death. Gene positive patients carry a pathogenic variant in the ATP1A3 -encoded Na/K ATPase alpha 3 isoform (ATP1A3), and the variant D801N is the most common pathogenic variant. Our group recently found that AHC patients with ATP1A3-D801N missense have short QTc and are at risk of ventricular fibrillation. Hypothesis: We hypothesized that the D801N variant results in reduced ATPase function leading to Ca 2+ overload resulting in shortened repolarization time and increased arrhythmogenic risk. Methods: We differentiated iPSCs into cardiomyocytes from an AHC patient hosting the ATP1A3-D801N variant (iPSC-CM D801N ), and an ostensibly healthy individual (iPSC-CM WT ). Patch clamp was conducted to determine APD90, and the ratio of cells with delayed after depolarization events (DADs). iPSC-CM WT were perfused with 10 -7 M ouabain, an ATP1A3 inhibitor, and APD90 and DADs were measured. SR store ([Ca 2+ ] SR ) was measured with Cal-520 dye under 10 mM caffeine. Resting [Ca 2+ ] i was measured with Fura-2 dye, and Na/Ca exchanger (NCX) peak current was measured as Ni 2+ sensitive current in a stepwise voltage-clamp protocol. Results: iPSC-CM D801N APD90 was shorter 40% than iPSC-CM WT . IPSC-CM WT treated with ouabain had 17% shorter APD90 than vehicle. iPSC-CM D801N had DADs (3/11 cells) when iPSC-CM WT (0/24) did not, and 6/12 ouabain treated iPSC-CM WT had DADs vs vehicle treated (0/18). Post caffeine, iPSC-CM D801N had higher [Ca 2+ ] SR than iPSC-CM WT . Finally, iPSC-CM D801N had higher resting [Ca 2+ ] i than iPSC-CM WT and iPSC-CM D801N had higher peak NCX current density at +80mV but no difference at RMP. Conclusions: Our results show that both iPSC-CM D801N and ouabain-treated iPSC-CM WT , have a shorter APD compared to WT controls and exhibit DADs. This is associated with higher NCX-mediated influx of Ca 2+ and increased intracellular Ca 2+ . Our findings highlight the emerging role of ATP1A3-D801N in creating a myocardium vulnerable to arrhythmias.

Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1

Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Cav2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs.

A neurophysiological limit and its biogeographic correlations: Cold-induced spreading depolarization in tropical butterflies.

The physiology of insects is directly influenced by environmental temperature, and thermal tolerance is therefore intrinsically linked to their thermal niche and distribution. Understanding the mechanisms that limit insect thermal tolerance is crucial to predicting biogeography and range shifts. Recent studies on locusts and flies suggest that the critical thermal minimum (CTmin) follows from a loss of CNS function via a spreading depolarization. We hypothesized that other insect taxa share this phenomenon. Here we investigate whether spreading depolarization events occur in butterflies exposed to cold. Supporting our hypothesis, we find that exposure to stressful cold induced spreading depolarization in all 12 species tested. This reinforces the idea that spreading depolarization is a common mechanism underlying the insect CTmin. Furthermore, our results highlight how CNS function is tuned to match species' environments. Further research into the physiology underlying spreading depolarization will likely elucidate key mechanisms determining insect thermal tolerance and ecology.

Synapse-specific diversity of distinct postsynaptic GluN2 subtypes defines transmission strength in spinal lamina I.

The unitary postsynaptic response to presynaptic quantal glutamate release is the fundamental basis of excitatory information transfer between neurons. The view, however, of individual glutamatergic synaptic connections in a population as homogenous, fixed-strength units of neural communication is becoming increasingly scrutinized. Here, we used minimal stimulation of individual glutamatergic afferent axons to evoke single synapse resolution postsynaptic responses from central sensory lamina I neurons in an ex vivo adult rat spinal slice preparation. We detected unitary events exhibiting a NMDA receptor component with distinct kinetic properties across synapses conferred by specific GluN2 subunit composition, indicative of GluN2 subtype-based postsynaptic heterogeneity. GluN2A, 2A and 2B, or 2B and 2D synaptic predominance functioned on distinct lamina I neuron types to narrowly, intermediately, or widely tune, respectively, the duration of evoked unitary depolarization events from resting membrane potential, which enabled individual synapses to grade differentially depolarizing steps during temporally patterned afferent input. Our results lead to a model wherein a core locus of proteomic complexity prevails at this central glutamatergic sensory synapse that involves distinct GluN2 subtype configurations. These findings have major implications for subthreshold integrative capacity and transmission strength in spinal lamina I and other CNS regions.

Predicting mitochondrial fission, fusion and depolarisation event locations from a single z-stack.

This paper documents the development of a novel method to predict the occurrence and exact locations of mitochondrial fission, fusion and depolarisation events in three dimensions. This novel implementation of neural networks to predict these events using information encoded only in the morphology of the mitochondria eliminate the need for time-lapse sequences of cells. The ability to predict these morphological mitochondrial events using a single image can not only democratise research but also revolutionise drug trials. The occurrence and location of these events were successfully predicted with a three-dimensional version of the Pix2Pix generative adversarial network (GAN) as well as a three-dimensional adversarial segmentation network called the Vox2Vox GAN. The Pix2Pix GAN predicted the locations of mitochondrial fission, fusion and depolarisation events with accuracies of 35.9%, 33.2% and 4.90%, respectively. Similarly, the Vox2Vox GAN achieved accuracies of 37.1%, 37.3% and 7.43%. The accuracies achieved by the networks in this paper are too low for the immediate implementation of these tools in life science research. They do however indicate that the networks have modelled the mitochondrial dynamics to some degree of accuracy and may therefore still be helpful as an indication of where events might occur if time lapse sequences are not available. The prediction of these morphological mitochondrial events have, to our knowledge, never been achieved before in literature. The results from this paper can be used as a baseline for the results obtained by future work.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

Detection of spreading depolarization events and spatiotemporal analysis for advancing stroke therapy.

While the presence of spreading depolarization (SD) and associated spreading depression have been well studied and known to be associated with post-ischemic brain damage, the spatiotemporal spread of these events from the site of injury is not well understood. With the recent development of high-density micro-electrocorticographic (ECoG) electrode arrays, monitoring the spread of the depolarizing events and associated depression is possible. The goal of this work is to define the electrocorticographic features of SD and associated depression across the multichannel array and search for patterns in these features that emerge across both space and time. We present the spatial distribution of features found from chronic ECoG recordings acquired from awake behaving rats induced with a rodent model of stroke. SD events were detected with an unsupervised algorithm that searched for a stereotyped pattern in the first derivative of the ECoG. The algorithm yielded a 58% correct detection rate on average across four rats, and a 36% false positive rate. We defined key electrophysiological features and mapped them onto the physical brain regions using MATLAB, such as the peak-to-peak amplitude of each SD event, the width (or duration) of the SD event, direct current (DC) level, and average rate of decline in the signal baseline. We performed k-means clustering to the activity in this feature space which yielded three contiguous regions in physical space. The elbow optimization method was applied to a distortion metric and indicated that 3 clusters was optimal. These findings motivate us to conduct future studies that would verify whether these 3 clusters in electrode-space correspond to immunohistochemically defined regions of tissue health, namely, infarct, penumbra, and healthy tissue. Clinical Relevance- The extent and severity of damage that stroke ultimately causes is suspected to be related to the progression of spreading depolarization and associated depression. An understanding of how the features of these electrophysiological events progress across the brain and over time is an important step toward eventual development of closed-loop therapies which limit and minimize the long-term effects of stroke.

Postischemic Neuroprotection of Aminoethoxydiphenyl Borate Associates Shortening of Peri-Infarct Depolarizations.

Brain stroke is a highly prevalent pathology and a main cause of disability among older adults. If not promptly treated with recanalization therapies, primary and secondary mechanisms of injury contribute to an increase in the lesion, enhancing neurological deficits. Targeting excitotoxicity and oxidative stress are very promising approaches, but only a few compounds have reached the clinic with relatively good positive outcomes. The exploration of novel targets might overcome the lack of clinical translation of previous efficient preclinical neuroprotective treatments. In this study, we examined the neuroprotective properties of 2-aminoethoxydiphenyl borate (2-APB), a molecule that interferes with intracellular calcium dynamics by the antagonization of several channels and receptors. In a permanent model of cerebral ischemia, we showed that 2-APB reduces the extent of the damage and preserves the functionality of the cortical territory, as evaluated by somatosensory evoked potentials (SSEPs). While in this permanent ischemia model, the neuroprotective effect exerted by the antioxidant scavenger cholesteronitrone F2 was associated with a reduction in reactive oxygen species (ROS) and better neuronal survival in the penumbra, 2-APB did not modify the inflammatory response or decrease the content of ROS and was mostly associated with a shortening of peri-infarct depolarizations, which translated into better cerebral blood perfusion in the penumbra. Our study highlights the potential of 2-APB to target spreading depolarization events and their associated inverse hemodynamic changes, which mainly contribute to extension of the area of lesion in cerebrovascular pathologies.

A computational model of organism development and carcinogenesis resulting from cells’ bioelectric properties and communication

A sound theory of biological organization is clearly missing for a better interpretation of observational results and faster progress in understanding life complexity. The availability of such a theory represents a fundamental progress in explaining both normal and pathological organism development. The present work introduces a computational implementation of some principles of a theory of organism development, namely that the default state of cells is proliferation and motility, and includes the principle of variation and organization by closure of constraints. In the present model, the bioelectric context of cells and tissue is the field responsible for organization, as it regulates cell proliferation and the level of communication driving the system’s evolution. Starting from a depolarized (proliferative) cell, the organism grows to a certain size, limited by the increasingly polarized state after successive proliferation events. The system reaches homeostasis, with a depolarized core (proliferative cells) surrounded by a rim of polarized cells (non-proliferative in this condition). This state is resilient to cell death (random or due to injure) and to limited depolarization (potentially carcinogenic) events. Carcinogenesis is introduced through a localized event (a spot of depolarized cells) or by random depolarization of cells in the tissue, which returns cells to their initial proliferative state. The normalization of the bioelectric condition can reverse this out-of-equilibrium state to a new homeostatic one. This simplified model of embryogenesis, tissue organization and carcinogenesis, based on non-excitable cells’ bioelectric properties, can be made more realistic with the introduction of other components, like biochemical fields and mechanical interactions, which are fundamental for a more faithful representation of reality. However, even a simple model can give insight for new approaches in complex systems and suggest new experimental tests, focused in its predictions and interpreted under a new paradigm.

Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes.

Simple SummaryIn this study, the induced pluripotent stem cell-derived cardiomyocyte model from a patient with long QT syndrome harboring a heterozygous Nav1.5 R1623Q mutation exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). FPDcF was shortened with mexiletine treatment and increased the frequency of arrhythmia-like EAD events following E4031, an Ikr blocker, administration. These characteristics partly reflect the patient phenotypes. As the R1623Q mutation is related to severe congenital LQT syndrome in fetuses and neonates, the effect of the neonatal variants on the electrophysiological properties of the R1623Q mutant was examined using an automated patch-clamp system. Our results demonstrated that both R1623Q and neonatal R1623Q delayed inactivation of INa and increased late Na current. We speculated that neonatal Nav1.5 ameliorates QTc prolongation. Developmental switching of neonatal/adult Nav1.5 isoforms might play a role in the mechanisms underlying severe long QT syndrome in fetuses and neonates.The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult Nav1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.

Mitochondrial event localiser (MEL) to quantitativelydescribe fission, fusion and depolarisation in the three-dimensional space.

Mitochondrial fission and fusion play an important role not only in maintaining mitochondrial homeostasis but also in preserving overall cellular viability. However, quantitative analysis based on the three-dimensional localisation of these highly dynamic mitochondrial events in the cellular context has not yet been accomplished. Moreover, it remains largely uncertain where in the mitochondrial network depolarisation is most likely to occur. We present the mitochondrial event localiser (MEL), a method that allows high-throughput, automated and deterministic localisation and quantification of mitochondrial fission, fusion and depolarisation events in large three-dimensional microscopy time-lapse sequences. In addition, MEL calculates the number of mitochondrial structures as well as their combined and average volume for each image frame in the time-lapse sequence. The mitochondrial event locations can subsequently be visualised by superposition over the fluorescence micrograph z-stack. We apply MEL to both control samples as well as to cells before and after treatment with hydrogen peroxide (H2O2). An average of 9.3/7.2/2.3 fusion/fission/depolarisation events per cell were observed respectively for every 10 sec in the control cells. With peroxide treatment, the rate initially shifted toward fusion with and average of 15/6/3 events per cell, before returning to a new equilibrium not far from that of the control cells, with an average of 6.2/6.4/3.4 events per cell. These MEL results indicate that both pre-treatment and control cells maintain a fission/fusion equilibrium, and that depolarisation is higher in the post-treatment cells. When individually validating mitochondrial events detected with MEL, for a representative cell for the control and treated samples, the true-positive events were 47%/49%/14% respectively for fusion/fission/depolarisation events. We conclude that MEL is a viable method of quantitative mitochondrial event analysis.