A computational model to uncover the biophysical underpinnings of neural firing heterogeneity in dissociated hippocampal cultures.

Calcium (Ca2+ ) imaging reveals a variety of correlated firing in cultures of dissociated hippocampal neurons, pinpointing the non-synaptic paracrine release of glutamate as a possible mediator for such firing patterns, although the biophysical underpinnings remain unknown. An intriguing possibility is that extracellular glutamate could bind metabotropic receptors linked with inositol trisphosphate (IP3 ) mediated release of Ca2+ from the endoplasmic reticulum of individual neurons, thereby modulating neural activity in combination with sarco/endoplasmic reticulum Ca2+ transport ATPase (SERCA) and voltage-gated Ca2+ channels (VGCC). However, the possibility that such release may occur in different neuronal compartments and can be inherently stochastic poses challenges in the characterization of such interplay between various Ca2+ channels. Here we deploy biophysical modeling in association with Monte Carlo parameter sampling to characterize such interplay and successfully predict experimentally observed Ca2+ patterns. The results show that the neurotransmitter level at the plasma membrane is the extrinsic source of heterogeneity in somatic Ca2+ transients. Our analysis, in particular, identifies the origin of such heterogeneity to an intrinsic differentiation of hippocampal neurons in terms of multiple cellular properties pertaining to intracellular Ca2+ signaling, such as VGCC, IP3 receptor, and SERCA expression. In the future, the biophysical model and parameter estimation approach used in this study can be upgraded to predict the response of a system of interconnected neurons.

Hippocampal dendritic spines express the RyR3 but not the RyR2 ryanodine receptor isoform.

The hippocampus is a brain region implicated in synaptic plasticity and memory formation; both processes require neuronal Ca2+ signals generated by Ca2+ entry via plasma membrane Ca2+ channels and Ca2+ release from the endoplasmic reticulum (ER). Through Ca2+-induced Ca2+ release, the ER-resident ryanodine receptor (RyR) Ca2+ channels amplify and propagate Ca2+ entry signals, leading to activation of cytoplasmic and nuclear Ca2+-dependent signaling pathways required for synaptic plasticity and memory processes. Earlier reports have shown that mice and rat hippocampus expresses mainly the RyR2 isoform, with lower expression levels of the RyR3 isoform and almost undetectable levels of the RyR1 isoform; both the RyR2 and RyR3 isoforms have central roles in synaptic plasticity and hippocampal-dependent memory processes. Here, we describe that dendritic spines of rat primary hippocampal neurons express the RyR3 channel isoform, which is also expressed in the neuronal body and neurites. In contrast, the RyR2 isoform, which is widely expressed in the neuronal body and neurites of primary hippocampal neurons, is absent from the dendritic spines. We propose that this asymmetric distribution is of relevance for hippocampal neuronal function. We suggest that the RyR3 isoform amplifies activity-generated Ca2+ entry signals at postsynaptic dendritic spines, from where they propagate to the dendrite and activate primarily RyR2-mediated Ca2+ release, leading to Ca2+ signal propagation into the soma and the nucleus where they activate the expression of genes that mediate synaptic plasticity and memory.

Spontaneous Ca2+ events are linked to the development of neuronal firing during maturation in mice primary hippocampal culture cells.

Calcium is one of the most vital intracellular secondary messengers that tightly regulates a variety of cell physiology processes, especially in the brain. Using a fluorescent Ca2+-sensitive Oregon Green probe, we revealed three different amplitude distributions of spontaneous Ca2+ events (SCEs) in neurons between 15 and 26 days in vitro (DIV) culture maturation. We detected a series of amplitude events: micro amplitude SCE (microSCE) 25% increase from the baseline, intermediate amplitude SCE (interSCE) as 25-75%, and macro amplitude SCE (macroSCE) - over 75%. The SCEs were fully dependent on extracellular Ca2+ and neuronal network activity and vanished in the Ca2+-free solution, 10 mM Mg2+-block, or in the presence of voltage-gated Na+-channel blocker, tetrodotoxin. Combined patch-clamp and Ca2+-imaging techniques revealed that microSCE match single action potential (AP), interSCE - burst of 3-12 APs, and macroSCE - 'superburst' of 10+ APs. MicroSCEs were blocked by a common α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) receptor antagonist, CNQX. The γ-aminobutyric acid (GABA) A-type receptor (GABAAR) picrotoxin blockade and L-type voltage-dependent Ca2+-channel inhibitor diltiazem significantly reduced microSCE frequency. InterSCEs were inhibited by CNQX, but picrotoxin treatment significantly increased its amplitude. The N-methyl-d-aspartate (NMDA) receptor antagonist, D-APV, voltage-gated K+-channel blocker, tetraethylammonium, noticeably suppressed interSCE amplitude. We also demonstrate that macroSCEs were AMPA/KA receptor-independent.

Inhibition of Neuronal Necroptosis Mediated by RIPK1 Provides Neuroprotective Effects on Hypoxia and Ischemia In Vitro and In Vivo.

Ischemic brain injury is a widespread pathological condition, the main components of which are a deficiency of oxygen and energy substrates. In recent years, a number of new forms of cell death, including necroptosis, have been described. In necroptosis, a cascade of interactions between the kinases RIPK1 and RIPK3 and the MLKL protein leads to the formation of a specialized death complex called the necrosome, which triggers MLKL-mediated destruction of the cell membrane and necroptotic cell death. Necroptosis probably plays an important role in the development of ischemia/reperfusion injury and can be considered as a potential target for finding methods to correct the disruption of neural networks in ischemic damage. In the present study, we demonstrated that blockade of RIPK1 kinase by Necrostatin-1 preserved the viability of cells in primary hippocampal cultures in an in vitro model of glucose deprivation. The effect of RIPK1 blockade on the bioelectrical and metabolic calcium activity of neuron-glial networks in vitro using calcium imaging and multi-electrode arrays was assessed for the first time. RIPK1 blockade was shown to partially preserve both calcium and bioelectric activity of neuron-glial networks under ischemic factors. However, it should be noted that RIPK1 blockade does not preserve the network parameters of the collective calcium dynamics of neuron-glial networks, despite the maintenance of network bioelectrical activity (the number of bursts and the number of spikes in the bursts). To confirm the data obtained in vitro, we studied the effect of RIPK1 blockade on the resistance of small laboratory animals to in vivo modeling of hypoxia and cerebral ischemia. The use of Necrostatin-1 increases the survival rate of C57BL mice in modeling both acute hypobaric hypoxia and ischemic brain damage.

The STIM1/2-Regulated Calcium Homeostasis Is Impaired in Hippocampal Neurons of the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of age-related dementia. Neuronal calcium homeostasis impairment may contribute to AD. Here we demonstrated that voltage-gated calcium (VGC) entry and store-operated calcium (SOC) entry regulated by calcium sensors of intracellular calcium stores STIM proteins are affected in hippocampal neurons of the 5xFAD transgenic mouse model. We observed excessive SOC entry in 5xFAD mouse neurons, mediated by STIM1 and STIM2 proteins with increased STIM1 contribution. There were no significant changes in cytoplasmic calcium level, endoplasmic reticulum (ER) bulk calcium levels, or expression levels of STIM1 or STIM2 proteins. The potent inhibitor BTP-2 and the FDA-approved drug leflunomide reduced SOC entry in 5xFAD neurons. In turn, excessive voltage-gated calcium entry was sensitive to the inhibitor of L-type calcium channels nifedipine but not to the T-type channels inhibitor ML218. Interestingly, the depolarization-induced calcium entry mediated by VGC channels in 5xFAD neurons was dependent on STIM2 but not STIM1 protein in cells with replete Ca2+ stores. The result gives new evidence on the VGC channel modulation by STIM2. Overall, the data demonstrate the changes in calcium signaling of hippocampal neurons of the AD mouse model, which precede amyloid plaque accumulation or other signs of pathology manifestation.

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro.

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.

Regulation of synaptic scaling by action potential-independent miniature neurotransmission.

Synaptic scaling represents a homeostatic adjustment in synaptic strength that was first identified as a cell-wide mechanism to achieve firing rate homeostasis after perturbations to spiking activity levels. In this review, we consider a form of synaptic scaling that is triggered by changes in action potential-independent neurotransmitter release. This plasticity appears to be both triggered and expressed locally at the dendritic site of the synapse that experiences a perturbation. A discussion of different forms of scaling triggered by different perturbations is presented. We consider work from multiple groups supporting this form of scaling, which we call neurotransmission-based scaling. This class of homeostatic synaptic plasticity is compared in studies using hippocampal and cortical cultures, as well as in vivo work in the embryonic chick spinal cord. Despite differences in the tissues examined, there are clear similarities in neurotransmission-based scaling, which appear to be molecularly distinct from the originally described spike-based scaling.

ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15 min followed by 24 h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1 µg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1 µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70 mM K+. A sub effective concentration of ConBr (0.01 µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3 µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Formation of Neural Networks in 3D Scaffolds Fabricated by Means of Laser Microstereolithography.

We developed and tested new 3D scaffolds for neurotransplantation. Scaffolds of predetermined architectonic were prepared using microstereolithography technique. Scaffolds were highly biocompatible with the nervous tissue cells. In vitro studies showed that the material of fabricated scaffolds is not toxic for dissociated brain cells and promotes the formation of functional neural networks in the matrix. These results demonstrate the possibility of fabrication of tissue-engineering constructs for neurotransplantation based on created scaffolds.

Different pools of postsynaptic GABAA receptors mediate inhibition evoked by low- and high-frequency presynaptic stimulation at hippocampal synapses.

Patterns of short-term synaptic plasticity could considerably differ between synapses of the same axon. This may lead to separation of synaptic receptors transmitting either low- or high-frequency signals and, therefore, may have functional consequences for the information transfer in the brain. Here, we estimated a degree of such separation at hippocampal GABAergic synapses using a use-dependent GABAA receptor antagonist, picrotoxin, to selectively suppress a pool of GABAA receptors monosynaptically activated during the low-frequency stimulation. The relative changes in postsynaptic responses evoked by the high-frequency stimulation before and after such block were used to estimate the contribution of this GABAA receptor pool to synaptic transmission at high frequencies. Using this approach, we have shown that IPSCs evoked by low-frequency (0.2 Hz) stimulation and asynchronous currents evoked by high-frequency (20-40 Hz) stimulation are mediated by different pools of postsynaptic GABAA receptors. Thus, our findings suggest that inhibition produced by a single hippocampal interneuron may be selectively routed to different postsynaptic targets depending on the presynaptic discharge frequency.

Fluorescence lifetime imaging of AMPA receptor endocytosis in living neurons: effects of Aß and PP1.

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aß) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aß triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aß exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.

Oxygen-Glucose Deprivation in Organotypic Hippocampal Cultures Leads to Cytoskeleton Rearrangement and Immune Activation: Link to the Potential Pathomechanism of Ischaemic Stroke.

Ischaemic stroke is characterized by a sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. As a result of this process, neurons in the ischaemic core are deprived of oxygen and trophic substances and are consequently destroyed. Tissue damage in brain ischaemia results from a complex pathophysiological cascade comprising various distinct pathological events. Ischaemia leads to brain damage by stimulating many processes, such as excitotoxicity, oxidative stress, inflammation, acidotoxicity, and apoptosis. Nevertheless, less attention has been given to biophysical factors, including the organization of the cytoskeleton and the mechanical properties of cells. Therefore, in the present study, we sought to evaluate whether the oxygen-glucose deprivation (OGD) procedure, which is a commonly accepted experimental model of ischaemia, could affect cytoskeleton organization and the paracrine immune response. The abovementioned aspects were examined ex vivo in organotypic hippocampal cultures (OHCs) subjected to the OGD procedure. We measured cell death/viability, nitric oxide (NO) release, and hypoxia-inducible factor 1α (HIF-1α) levels. Next, the impact of the OGD procedure on cytoskeletal organization was evaluated using combined confocal fluorescence microscopy (CFM) and atomic force microscopy (AFM). Concurrently, to find whether there is a correlation between biophysical properties and the immune response, we examined the impact of OGD on the levels of crucial ischaemia cytokines (IL-1ß, IL-6, IL-18, TNF-α, IL-10, IL-4) and chemokines (CCL3, CCL5, CXCL10) in OHCs and calculated Pearsons' and Spearman's rank correlation coefficients. The results of the current study demonstrated that the OGD procedure intensified cell death and nitric oxide release and led to the potentiation of HIF-1α release in OHCs. Moreover, we presented significant disturbances in the organization of the cytoskeleton (actin fibers, microtubular network) and cytoskeleton-associated protein 2 (MAP-2), which is a neuronal marker. Simultaneously, our study provided new evidence that the OGD procedure leads to the stiffening of OHCs and a malfunction in immune homeostasis. A negative linear correlation between tissue stiffness and branched IBA1 positive cells after the OGD procedure suggests the pro-inflammatory polarization of microglia. Moreover, the negative correlation of pro- and positive anti-inflammatory factors with actin fibers density indicates an opposing effect of the immune mediators on the rearrangement of cytoskeleton induced by OGD procedure in OHCs. Our study constitutes a basis for further research and provides a rationale for integrating biomechanical and biochemical methods in studying the pathomechanism of stroke-related brain damage. Furthermore, presented data pointed out the interesting direction of proof-of-concept studies, in which follow-up may establish new targets for brain ischemia therapy.

Amyloid ß-Oligomers Inhibit the Nuclear Ca2+ Signals and the Neuroprotective Gene Expression Induced by Gabazine in Hippocampal Neurons.

Hippocampal neuronal activity generates dendritic and somatic Ca2+ signals, which, depending on stimulus intensity, rapidly propagate to the nucleus and induce the expression of transcription factors and genes with crucial roles in cognitive functions. Soluble amyloid-beta oligomers (AßOs), the main synaptotoxins engaged in the pathogenesis of Alzheimer's disease, generate aberrant Ca2+ signals in primary hippocampal neurons, increase their oxidative tone and disrupt structural plasticity. Here, we explored the effects of sub-lethal AßOs concentrations on activity-generated nuclear Ca2+ signals and on the Ca2+-dependent expression of neuroprotective genes. To induce neuronal activity, neuron-enriched primary hippocampal cultures were treated with the GABAA receptor blocker gabazine (GBZ), and nuclear Ca2+ signals were measured in AßOs-treated or control neurons transfected with a genetically encoded nuclear Ca2+ sensor. Incubation (6 h) with AßOs significantly reduced the nuclear Ca2+ signals and the enhanced phosphorylation of cyclic AMP response element-binding protein (CREB) induced by GBZ. Likewise, incubation (6 h) with AßOs significantly reduced the GBZ-induced increases in the mRNA levels of neuronal Per-Arnt-Sim domain protein 4 (Npas4), brain-derived neurotrophic factor (BDNF), ryanodine receptor type-2 (RyR2), and the antioxidant enzyme NADPH-quinone oxidoreductase (Nqo1). Based on these findings we propose that AßOs, by inhibiting the generation of activity-induced nuclear Ca2+ signals, disrupt key neuroprotective gene expression pathways required for hippocampal-dependent learning and memory processes.

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures.

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON's considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior.

MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures.

Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures is an on-going question. Here, we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines.

The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Improves Ex Vivo and In Vivo Amyloid Beta (1-42)-Induced Neuroinflammation in Mouse Models of Alzheimer's Disease.

The major histopathological hallmarks of Alzheimer's disease (AD) include ß-amyloid (Aß) plaques, neurofibrillary tangles, and neuronal loss. Aß 1-42 (Aß1-42) has been shown to induce neurotoxicity and secretion of proinflammatory mediators that potentiate neurotoxicity. Proinflammatory and neurotoxic activities of Aß1-42 were shown to be mediated by interactions with several cell surface receptors, including the chemotactic G protein-coupled N-formyl peptide receptor 2 (FPR2). The present study investigated the impact of a new FPR2 agonist, MR-39, on the neuroinflammatory response in ex vivo and in vivo models of AD. To address this question, organotypic hippocampal cultures from wild-type (WT) and FPR2-deficient mice (knockout, KO, FPR2-/-) were treated with fibrillary Aß1-42, and the effect of the new FPR2 agonist MR-39 on the release of pro- and anti-inflammatory cytokines was assessed. Similarly, APP/PS1 double-transgenic AD mice were treated for 20 weeks with MR-39, and immunohistological staining was performed to assess neuronal loss, gliosis, and Aß load in the hippocampus and cortex. The data indicated that MR-39 was able to reduce the Aß1-42-induced release of proinflammatory cytokines and to improve the release of anti-inflammatory cytokines in mouse hippocampal organotypic cultures. The observed effect was apparently related to the inhibition of the MyD88/TRAF6/NFкB signaling pathway and a decrease in NLRP3 inflammasome activation. Administration of MR-39 to APP/PS1 mice improved neuronal survival and decreased microglial cell density and plaque load.These results suggest that FPR2 may be a promising target for alleviating the inflammatory process associated with AD and that MR-39 may be a useful therapeutic agent for AD.

Comparative Analysis of the Effects of Upconversion Nanoparticles on Normal and Tumor Brain Cells.

Glioma is the most aggressive type of brain tumors encountered in medical practice. The high frequency of diagnosed cases and risk of metastasis, the low efficiency of traditional therapy, and the usually unfavorable prognosis for patients dictate the need to develop alternative or combined approaches for an early diagnosis and treatment of this pathology. High expectations are placed on the use of upconversion nanoparticles (UCNPs). In this study, we have produced and characterized UCNPs doped with the rare-earth elements ytterbium and thulium. Our UCNPs had photoluminescence emission maxima in the visible and infrared spectral regions, which allow for deep optical imaging of tumor cells in the brain. Moreover, we evaluated the toxicity effects of our UCNPs on a normal brain and glioma cells. It was revealed that our UCNPs are non-toxic to glioma cells but have a moderate cytotoxic effect on primary neuronal cultures at high concentrations, a condition that is characterized by a decreased cellular viability and changes in the functional metabolic activity of neuron-glial networks. Despite the great potential associated with the use of these UCNPs as fluorescent markers, there is a need for further studies on the rate of the UCNPs accumulation and excretion in normal and tumor brain cells, and the use of their surface modifications in order to reduce their cytotoxic effects.

Variability of seizure-like activity in an in vitro model of epilepsy depends on the electrical recording method.

BACKGROUND: Hippocampal and cortical slice-based models are widely used to study seizures and epilepsy. Seizure detection and quantification are essential components for studying mechanisms of epilepsy and assessing therapeutic interventions. To obtain meaningful signals and maximize experimental throughput, variability should be minimized. Some electrical recording methods require insertion of an electrode into neuronal tissue, change in slice chemical microenvironment, and transients in temperature and pH. These perturbations can cause acute and long-term alterations of the neuronal network which may be reflected in the variability of the recorded signal. NEW METHOD: In this study we investigated the effect of experimental perturbations in three local field potential (LFP) recording methods including substrate micro-wires (s-MWs), multiple electrode arrays (MEAs), and inserted micro wire electrodes (i-MW). These methods enabled us to isolate effects of different perturbations. We used organotypic hippocampal slices (OHCs) as an in-vitro model of posttraumatic epilepsy. To investigate the effect of the disturbances caused by the recording method on the paroxysmal events, we introduced jitter analysis, which is sensitive to small differences in the seizure spike timing. RESULTS: Medium replacement can introduce long-lasting perturbations. Electrode insertion increased variability on a shorter time scale. OHCs also underwent spontaneous state transitions characterized by transient increases in variability. COMPARISON WITH EXISTING METHODS: This new method of seizure waveform analysis allows for more sensitive assessment of variability of ictal events than simply measuring seizure frequency and duration. CONCLUSION: We demonstrated that some of the variability in OHC recordings are due to experimental perturbations while some are spontaneous and independent of recording method.

Neuroprotective Effect of HIF Prolyl Hydroxylase Inhibition in an In Vitro Hypoxia Model.

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 µM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 µÐœ and 15 µÐœ neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 µÐœ, but not for 1 µÐœ neuradapt. Network connectivity is better preserved with immediate treatment using 1 µÐœ neuradapt than with 15 µÐœ, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 µÐœ and functional activity at 15 µÐœ. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.