Spontaneous Bioelectric Activity Research Articles

Role of RIPK1 kinase in neuronal-glial network adaptation under hypoxic conditions

Cerebral hypoxia is a condition characterized by a reduced oxygen supply to tissues that plays a crucial role in the pathogenesis of numerous neurodegenerative diseases. In hypoxia, intracellular signaling cascades are activated, ultimately leading to various forms of nerve cell death. The initiation of necroptosis under hypoxic conditions is governed by PIPK1 kinase, and its inhibition could potentially offer neuroprotection against hypoxic damage [1–3]. Studies investigating the effects of blocking RIPK1 kinase on the activity of neuronal-glial networks are currently lacking. Consequently, RIPK1 kinase constitutes a promising objective for further exploration. Thus, the aim of this work is to examine the part played by RIPK1 kinase in the adjustment of neuronal-glial networks amid hypoxia. The study focused on primary cultures of nerve cells in the hippocampus of mouse embryos belonging to the C57Bl/6 line. Hypoxia was induced in vitro on the 14th day of culturing the nerve cells. The RIPK1 kinase inhibitor was administered 20 minutes prior to, during, and after the hypoxia intervention. After 7 days of the stress induction, the calcium and bioelectrical activity of the neuron-glial networks were evaluated. Calcium activity was assessed via the Oregon Green 488 BAPTA-1, AM (Thermo Fisher Scientific, USA) using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). The experiments assessed total percentage of oscillating cells in culture, the frequency, and duration of calcium events. The analysis of bioelectrical activity was conducted using the MEA 60 multi-electrode arrays from Multichannel Systems. The registered signal from the arrays was processed through the MEAMAN algorithms in MATLAB (Certificate of State Registration of Computer Program No. 2012611190). The average number of small network packages and spikes was estimated. Under physiological conditions, spontaneous calcium activity is observed by the 21st day of neuron-glial network development. The percentage of cells exhibiting calcium events is 60.64 ± 3.68%, the frequency of calcium oscillations is 1.52±0.22 osc/min, and the duration is 9.63±0.75 s. During hypoxia modeling, the percentage of cells exhibiting calcium events decreased to 34.77±4.08%, and the frequency of calcium oscillations decreased to 0.64±0.08 osc/min. The inhibition of RIPK1 kinase maintains the percentage of cells exhibiting calcium events at the level of intact cultures, which is 60.38±3.4%. By the 21st day of culturing primary nerve cell cultures under physiological conditions, spontaneous bioelectrical activity forms. This is indicated by parameters such as the average number of small network clusters and spikes. Hypoxia modeling has a negative impact on the development of spontaneous bioelectrical activity. In the “Intact” group, the average number of small network packs was 36.12±4.27 packs per 10 minutes, while in the cell culture with hypoxia, it was 15.87±3.03 packs per 10 minutes. Additionally, the “Intact” group had an average of 90.22±12.32 spikes, while the cell culture with hypoxia had only 11.58±4.7 spikes. However, blocking RIPK1 kinase during hypoxia preserved the average number of small network packs (23,49±2,14 packs/10 min). Thus, inhibiting RIPK1 kinase under hypoxic conditions preserves the proportion of cells that exhibit spontaneous calcium events and partially preserves spontaneous bioelectrical activity.

The Role of P2Y6 Receptors in the Mechanisms of the Neuroprotective Effect of Citicoline.

Spontaneous bioelectrical activity of the brain and the duration of gasping were recorded in mice during modeling of global strangulation ischemia of the brain against the background of preventive administration of citicoline. The maximum neuroprotective effect of citicoline was observed when it was administered 60 min before the simulation of ischemia and was completely prevented by preliminary administration of a selective P2Y6 receptor antagonist MRS2578. The obtained experimental data attest to the leading role of receptor mechanisms in the implementation of neuroprotective activity of citicoline.

Toxic Effect of Streptozotocin on Cultured Mouse Hippocampal Neurons.

In primary dissociated hippocampal cell cultures from 18-day-old mouse embryos, streptozotocin in concentrations of 2-5 mM produced a dose-dependent cytotoxic effect on day 3 in vitro, whereas on day 11 of culturing, the neurons were resistant to streptozotocin. The neurons in the 3-day cultures were functionally immature, which was seen from their weak spontaneous bioelectric activity in the form of rare single action potentials; by day 11 of culturing, the neurons reached a high level of differentiation and their functional properties acquired a character of network burst activity. Thus, streptozotocin had the most pronounced cytotoxic effect on immature hippocampal neurons in vitro.

CHARACTERISTICS OF THE FUNCTIONAL STATE OF THE MAXILLODENTAL SYSTEM ACCORDING TO THE ANALYSIS OF BIOELECTRICAL ACTIVITY OF MUSCLES IN PATIENTS WITH ASSOCIATED OCCLUSAL ABNORMALITIES WHICH REQUIRE COMPREHENSIVE TREATMENT

Malocclusions that have not been eliminated in time can cause complications such as periodontal diseases, TMJ and muscle dysfunctions.An objective indicator of functional disorders is the change of bioelectrical activity of masticatory muscles. The aim of our study was to estimate thefunctional state of maxillodental system in order to develop the individualized treatment and achievement of high functional and aestheticoptimum in patients with associated occlusal abnormalities who need complex treatment. All examined patients had deviations of the averageamplitude of bioelectrical activity of muscles, asymmetry in muscle function, bursts of spontaneous bioelectrical activity at rest, chewing cyclefragmentations, changes in rest and activity ratio during chewing. One of the most important criteria for the transition to the next stage of treatment isnormalization of bioelectrical activity of the muscles. Such approach allows to achieve stable and long-lasting functional results of the treatment.

Study of spontaneous bioelectrical activity of two hierarchically connected neural networks in vitro

Synchronization of neuronal subpopulations and spiking pattern propagation are essential features for bioelectrical activity of the brain. In this study, we proposed an engineering method to grow hierarchically connected networks in vitro through the microchannels and investigated the relations of local spiking propagation through the microchannels with integrated bursting events in the chambers.

Brain-Derived Neurotrophic Factor (BDNF) Preserves the Functional Integrity of Neural Networks in the β-Amyloidopathy Model in vitro.

Alzheimer’s disease (AD) is a widespread chronic neurodegenerative pathology characterized by synaptic dysfunction, partial neuronal death, cognitive decline and memory impairments. The major hallmarks of AD are extracellular senile amyloid plaques formed by various types of amyloid proteins (Aβ) and the formation and accumulation of intracellular neurofibrillary tangles. However, there is a lack of relevant experimental models for studying changes in neural network activity, the features of intercellular signaling or the effects of drugs on the functional activity of nervous cells during AD development. In this work, we examined two experimental models of amyloidopathy using primary hippocampal cultures. The first model involves the embryonic brains of 5xFAD mice; the second uses chronic application of amyloid beta 1-42 (Aβ1-42). The model based on primary hippocampal cells obtained from 5xFAD mice demonstrated changes in spontaneous network calcium activity characterized by a decrease in the number of cells exhibiting Ca2+ activity, a decrease in the number of Ca2+ oscillations and an increase in the duration of Ca2+ events from day 21 of culture development in vitro. Chronic application of Aβ1-42 resulted in the rapid establishment of significant neurodegenerative changes in primary hippocampal cultures, leading to marked impairments in neural network calcium activity and increased cell death. Using this model and multielectrode arrays, we studied the influence of amyloidopathy on spontaneous bioelectrical neural network activity in primary hippocampal cultures. It was shown that chronic Aβ application decreased the number of network bursts and spikes in a burst. The spatial structure of neural networks was also disturbed that characterized by reduction in both the number of key network elements (hubs) and connections between network elements. Moreover, application of brain-derived neurotrophic factor (BDNF) recombinant protein and BDNF hyperexpression by an adeno-associated virus vector partially prevented these amyloidopathy-induced neurodegenerative phenomena. BDNF maintained cell viability and spontaneous bioelectrical and calcium network activity in primary hippocampal cultures.

Changes in the morphofunctional development of the neuronal network in a dissociated cell culture of rat cerebral cortical neurons

Introduction. Study of the morphofunctional neuronal development in a dissociated cerebrocortical cell culture, using modern cell technologies, is a priority in experimental neurology, which is required for successful in vitro modelling of acute and chronic forms of cerebral pathology. Aim. A morphofunctional study of the in vitro changes in neuronal differentiation of rat cerebral cortical neurons, using a range of analysis methods, including immunohistochemistry, fluorescence, and electrophysiology. Materials and methods. We investigated the degree of culture differentiation on day 3–4 and day 10–11 of in vitro cultivation, measured by the intensity of the PSA-NCAM protein expression and the level of neuronal glutamate-induced calcium overload. That was then compared with the functional activity of the neuronal network cultivated on a microelectrode array, and with changes of the neuronal network’s activity in response to glutamate receptor overstimulation. Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 10–11 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 3–4 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 10–11, with had no effect on the generation of single action potentials. At the same time, kainate, the exogenous selective agonist of the one of the glutamate subtypes, completely blocked spontaneous activity of the mature neurons. Conclusion. Neocortical rat neurons reach the differentiation level necessary for the modelling of the cerebral pathologies by day 10–11 of in vitro cultivation. At this point, the process of disruption of the microelectrode array cultivated neuronal network by the glutamate receptor overactivation, has become multilayered: excitotoxic glutamate-induced damage produces selective disruption of neuronal burst activity, and with the greater cytotoxicity caused by kainate, spontaneous bioelectrical activity is completely blocked.

Dynamics of Measures of the Functional State of the Central Nervous System in Navy Operators after Long Work Cycles on the Background of Cytoflavin

Objectives. To assess the efficacy Cytoflavin in correcting functional status and work capacity among Navy operators after long work cycles. Results from studies of 25 Navy operators aged 27–42 years were analyzed after voyages (baseline) and after correctional measures using Cytoflavin. Operator status was evaluated using the Ruslov Questionnaire for the Formal Dynamic Properties of Individuality and EEG recordings. Results and conclusions. The data obtained here provide evidence that Cytoflavin has positive effects on the energy component of the functional state of Navy operators during the post-voyage period, increasing psychomotor ergicity and speed and optimizing spontaneous brain bioelectrical activity. Activation of measures of the energy potential, phasic activity, and emotional reactions by Cytoflavin were linked with processes expanding the reserve capacities of the CNS in Navy operators after prolonged work cycles.

Features of Neural Network Formation and Their Functions in Primary Hippocampal Cultures in the Context of Chronic TrkB Receptor System Influence.

Discovering the mechanisms underlying homeostatic regulation in brain neural network formation and stability processes is one of the most urgent tasks in modern neuroscience. Brain-derived neurotrophic factor (BDNF) and the tropomyosin-related kinase B (TrkB) receptor system have long been considered the main regulators of neuronal survival and differentiation. The elucidation of methods for studying neural network activity makes investigating the complex mechanisms underlying neural network structure reorganization during development and detecting new mechanisms for neuronal activity remodeling possible. In this in vitro study, we investigated the effects of chronic BDNF (the main TrkB stimulator) and ANA-12 (a TrkB receptor system blocker) administration on the formation of neural-glial networks. The formation of spontaneous bioelectrical activity and functional neural structure depend on TrkB receptors, and blocking TrkB receptors inhibits full bioelectrical activity development. Cross-correlation analysis demonstrated the decisive role of TrkB in the formation and “strengths” of activity centers. Even though an appropriate ANA-12 concentration is non-toxic to nerve cells, numerous cells in culture medium containing this reagent do not exhibit metabolic activity and are not functionally involved in signal transmission processes. Electron microscopy studies revealed that chronically influencing the TrkB receptor system significantly alters synaptic and mitochondrial apparatus capture in cells, and functional analysis of mitochondrial activity confirmed these findings. Because knowledge of interactions between TrkB-mediated regulation and the mitochondrial state under normal conditions is rather limited, data on these relationships are particularly interesting and require further investigation. Thus, we assume that the molecular cascades mediated by TrkB actively participate in the formation of functionally complete brain neural networks.

AAV-Syn-BDNF-EGFP Virus Construct Exerts Neuroprotective Action on the Hippocampal Neural Network during Hypoxia In Vitro.

Brain-derived neurotrophic factor (BDNF) is one of the key signaling molecules that supports the viability of neural cells in various brain pathologies, and can be considered a potential therapeutic agent. However, several methodological difficulties, such as overcoming the blood–brain barrier and the short half-life period, challenge the potential use of BDNF in clinical practice. Gene therapy could overcome these limitations. Investigating the influence of viral vectors on the neural network level is of particular interest because viral overexpression affects different aspects of cell metabolism and interactions between neurons. The present work aimed to investigate the influence of the adeno-associated virus (AAV)-Syn-BDNF-EGFP virus construct on neural network activity parameters in an acute hypobaric hypoxia model in vitro. Materials and methods. An adeno-associated virus vector carrying the BDNF gene was constructed using the following plasmids: AAV-Syn-EGFP, pDP5, DJvector, and pHelper. The developed virus vector was then tested on primary hippocampal cultures obtained from C57BL/6 mouse embryos (E18). Acute hypobaric hypoxia was induced on day 21 in vitro. Spontaneous bioelectrical and calcium activity of neural networks in primary cultures and viability tests were analysed during normoxia and during the posthypoxic period. Results. BDNF overexpression by AAV-Syn-BDNF-EGFP does not affect cell viability or the main parameters of spontaneous bioelectrical activity in normoxia. Application of the developed virus construct partially eliminates the negative hypoxic consequences by preserving cell viability and maintaining spontaneous bioelectrical activity in the cultures. Moreover, the internal functional structure, including the activation pattern of network bursts, the number of hubs, and the number of connections within network elements, is also partially preserved. BDNF overexpression prevents a decrease in the number of cells exhibiting calcium activity and maintains the frequency of calcium oscillations. Conclusion. This study revealed the pronounced antihypoxic and neuroprotective effects of AAV-Syn-BDNF-EGFP virus transduction in an acute normobaric hypoxia model.

Analysis of Age-Related Dynamics and Gender-Specific Characteristics of Spontaneous Bioelectrical Activity and Components of Auditory Evoked Potentials in Junior School Students Living in the Arctic Region of the Russian Federation

We studied neurophysiological characteristics of the age-related development in junior school students (7–8 and 10–11 years of age) living in the Arctic region of the Russian Federation. The background electroencephalograms (EEGs) were recorded during quiet wakefulness with the eyes closed and open, and event related potentials (ERP) were recorded during the passive perception of sound stimuli within the oddball paradigm in the group of children (33 subjects, 18 boys and 15 girls). A decrease of the latency period and the spatial rearrangement of mismatch negativity with an increase in the amplitude in the centrofrontal cortex have been revealed in the groups of children aged 10–11 years during the perception of a rare stimulus and a decrease of the latency period of the Р300 component in the central and parietal areas associated with the maturation of mechanisms for involuntary auditory attention. Age-specific differences in the components of auditory ERP (N1 and N2) have been shown during passive perception of rare and frequent sounds, which reflect the processes of the morphofunctional maturation of the brain cortex in healthy Arctic school students (an increase of the N1 component amplitude, a decrese of the amplitude and the latency period of the N2 component). The analysis of the background EEG characteristics has shown both the common features, such as a decrease with age of the EEG power in the Δ and θ bands in the eyes-open state, and the different direction and topographic specificities in the age-dependent reorganization of bioelectrical activity in boys and girls in the α1 and α2 EEG bands.

Intracerebral electroencephalography in targeting anterior thalamic nucleus for deep brain stimulation in refractory epilepsy

BackgroundResults of DBS of ATN in refractory epilepsy depend on accuracy of the electrode's location. We searched for characteristic intraoperative, intracerebral EEG recording pattern from anterior thalamic nuclei (ATNs) as a biological marker for verifying the electrode's position. MethodsThere were six patients with refractory epilepsy scheduled for deep brain stimulation (DBS) procedure. At surgery, to map the target, we recorded EEG from each lead of DBS electrodes. One patient underwent a 24 hours EEG with continuous recording from both ATNs before internalization of stimulator units. ResultsIn all patients we recorded spontaneous bioelectric activity of ATNs. The pattern of the recording from the ATN was similar in all cases. In the one patient where 24-hour recording was done with simultaneous scalp EEG, a complex partial seizure was captured. ConclusionThis is the first report of using DBS electrode for intraoperative EEG recordings from the ATN in patients with refractory epilepsy. Since we managed to find the characteristic pattern of bioelectric activity of ATN, this technique seems to be a promising method for targeting this structure during the operation.

Features of exogenous influence of the Brain-derived neurotrophic factor and blockade of TrkB receptor on the neural network activity in primary hippocampal cultures

Features of exogenous influence of the Brain-derived neurotrophic factor and blockade of TrkB receptor on the neural network activity in primary hippocampal cultures

Glial cell line-derived neurotrophic factor (GDNF) counteracts hypoxic damage to hippocampal neural network function in vitro

Glial cell line-derived neurotrophic factor (GDNF) is regarded as a potent neuroprotector and a corrector of neural network activity in stress conditions. This work aimed to investigate the effect of GDNF on primary hippocampal cultures during acute normobaric hypoxia. Hypoxia induction was performed using day 14 in vitro cultures derived from mouse embryos (E18) with the preventive addition of GDNF (1 ng/ml) to the culture medium 10 min before oxygen deprivation. An analysis of spontaneous bioelectrical activity that included defining the internal neural network structure, morphological studies, and viability tests was performed during the post-hypoxic period.This study revealed that GDNF does not influence spontaneous network activity during normoxia but protects cultures from cell death and maintains the network activity during hypoxia. GDNF created unique conditions that supported the viability of cells even in cases of cellular mitochondrial damage. GDNF partially negated the consequences of hypoxia by influencing synaptic plasticity. Intravital mRNA detection identified fewer GluR2 mRNA-positive cells, whereas GDNF preserved the number of these cells in the post-hypoxic period. Activation of the synthesis of GluR2 subunits of AMPA-receptors is one possible mechanism of the neuroprotective action of GDNF.

Glial cell line-derived neurotrophic factor as a new approach for functional neural networks reorganization in oxygen stress conditions

Glial cell line-derived neurotrophic factor as a new approach for functional neural networks reorganization in oxygen stress conditions

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

TrkB-Mediated Neuroprotective and Antihypoxic Properties of Brain-Derived Neurotrophic Factor.

The neuroprotective and antihypoxic effects of brain-derived neurotrophic factor (BDNF) on dissociated hippocampal cultures in a hypoxia model were investigated. These experiments demonstrate that 10 minutes of normobaric hypoxia increased the number of dead cells in primary culture, whereas a preventive application of BDNF increased the number of viable cells. Spontaneous bioelectrical and calcium activity in neural networks was analyzed using multielectrode arrays and functional intravital calcium imaging. The results indicate that BDNF affects the functional parameters of neuronal networks in dissociated hippocampal cultures over the 7-day posthypoxic period. In addition, the effects of k252a, an antagonist of tropomyosin-related kinase B (TrkB), on functional bioelectrical activity during and after acute hypoxia were investigated. It was shown that the protective effects of BDNF are associated with binding to the TrkB receptor. Finally, intravital fluorescent mRNA probes were used to study the role of NF-κB1 in the protective effects of BDNF. Our experiments revealed that BDNF application stimulates NF-κB1 mRNA synthesis in primary dissociated hippocampal cells under normal conditions but not in hypoxic state.

Modulation of network activity and induction of homeostatic synaptic plasticity by enzymatic removal of heparan sulfates.

Heparan sulfates (HSs) are complex and highly active molecules that are required for synaptogenesis and long-term potentiation. A deficit in HSs leads to autistic phenotype in mice. Here, we investigated the long-term effect of heparinase I, which digests highly sulfated HSs, on the spontaneous bioelectrical activity of neuronal networks in developing primary hippocampal cultures. We found that chronic heparinase treatment led to a significant reduction of the mean firing rate of neurons, particularly during the period of maximal neuronal activity. Furthermore, firing pattern in heparinase-treated cultures often appeared as epileptiform bursts, with long periods of inactivity between them. These changes in network activity were accompanied by an increase in the frequency and amplitude of miniature postsynaptic excitatory currents, which could be described by a linear up-scaling of current amplitudes. Biochemically, we observed an upregulation in the expression of the glutamate receptor subunit GluA1, but not GluA2, and a strong increase in autophosphorylation of α and β Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), without changes in the levels of kinase expression. These data suggest that a deficit in HSs triggers homeostatic synaptic plasticity and drastically affects functional maturation of neural network.

Effect of N-arachidonoyl dopamine on activity of neuronal network in primary hippocampus culture upon hypoxia modelling.

We studied the effect of endocannabinoid N-arachidonoyl dopamine on spontaneous bioelectric activity of cultured hippocampal neurons in a model of hypoxia/reoxygenation. Incubation under hypoxic conditions induced irreversible decrease in spontaneous bioelectric activity of neurons and their death. Application of N-arachidonoyl dopamine during hypoxia and in the post-hypoxic period preserved bioelectric activity and viability of neurons. The protective effect of N-arachidonoyl dopamine was primarily mediated by type I cannabinoid receptors.

Ouabain modulation of snail Br neuron bursting activity after the exposure to 10 mT static magnetic field revealed by Higuchi fractal dimension.

Aim of this study was to investigate the application of normalized mean of the empirical Higuchi fractal dimension (FD) distributions, as a new approach to analyze the spontaneous bioelectrical activity of garden snail (Helix pomatia) Br neuron. The effect of ouabain on modulation of Br neuron bursting activity before and after the exposure to 10 mT static magnetic field (SMF) was observed by analyzing the following parameters: action potential (AP), interspike interval (ISI) and interbursting interval (IBI) components. Normalized mean of the empirical FD distributions were formed for the following experimental conditions: Control 1, Ouabain 1, Control 2, SMF 2, ASMF 2, Control 3, SMF 3 and Ouabain ASMF 3. Our main results have shown that ouabain without SMF induced increase in participation of AP and a decrease in participation of IBI components compared to the first control condition. However, in the presence of 10 mT SMF, ouabain-induced changes of measured parameters of Br neuron activity were less pronounced compared to the third control condition. We have shown that normalized mean of the empirical FD distributions is a useful method for detecting the changes in AP, ISI, and IBI components of complex bursting activity in altered physiological states.