Organotypic Hippocampal Slice Research Articles

Cellular resolution contributions to ictal population signals.

The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. This work provides a critical link between single neuron activity and population measures of seizure activity.

Retraction to: Potential Mechanisms Linking Cholesterol to Alzheimer's Disease-like Pathology in Rabbit Brain, Hippocampal Organotypic Slices, and Skeletal Muscle.

Retraction to: Potential Mechanisms Linking Cholesterol to Alzheimer's Disease-like Pathology in Rabbit Brain, Hippocampal Organotypic Slices, and Skeletal Muscle.

Long-term Rapamycin Treatment Inhibit AKT Activity and Lower Intracellular Calcium Expression in Organotypic Hippocampal Slice Cultures Model of Epilepsy

Despite the development of anti-epilepsy drugs, drug-refractory epilepsy still becomes a challenging problem along with an increased incidence of epilepsy. To face that challenge and increase patients’ quality of life, treatment of epilepsy must effectively prevent epileptogenesis, not only symptomatic treatment. AKT signaling pathway was proven to have important roles in epilepsy through its function in the synaptic plasticity, neurogenesis, axon guidance, modulation of the glutamate transporter, and activation of the Ca2+ channel. AKT also activated mTOR signaling pathway as activator of mTORC1 and also effector of mTORC2. Several studies showed the ability of long-term rapamycin treatment to inhibit mTORC2. This study used organotypic hippocampal slice cultures (OHSC) and long-term rapamycin treatment was administered for 3, 5, 8, and 10 days at a dose of 20 nM after induction of epilepsy by low-Mg2+ medium administration for 40 minutes. Low-Mg2+ medium administration induced seizure activity in OHSC showed by significant increase in intracellular Ca2+expressionand also significantly increase AKT activity. After administration of long-term rapamycin treatment AKT activity and intracellular Ca2+expression were significantly reduced. The longer the treatment of rapamycin, the lower the AKT activity and intracellular Ca2+expression. Long-term rapamycin treatment has the potential to become a novel epilepsy drug through its ability to attenuate AKT activity and suppress the seizures proven by lower intracellular Ca2+expression.

Changes of synaptic vesicles in three-dimensional synapse models by treatment with umbelliferone in scopolamine-induced hippocampal injury model

The neuroprotective effects of umbelliferone (UMB) were visualized in three-dimensional (3D) images on vesicle density changes of organotypic hippocampal slice tissues (OHSCs) induced by scopolamine by high voltage electron microscopy. Observations revealed that the number of vesicles decreased in OHSCs induced by scopolamine, and UMB was found to inhibit scopolamine-induced reduction in vesicles, resulting in an increase in vesicle count. These 3D models provide valuable insight for understanding the increase of synapse vesicles in hippocampal tissues treated with UMB.

Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy.

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15μm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.

Kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside): a novel neuroprotective agent from Diospryros kaki against cerebral ischemia-induced brain injury.

Our previous study demonstrated neuroprotective and therapeutic effects of a standardized flavonoid extract from leaves of Diospyros kaki L.f. (DK) on middle cerebral artery occlusion-and-reperfusion (MCAO/R)-induced brain injury and its underlying mechanisms. This study aimed to clarify flavonoid components responsible for the effects of DK using in vitro and in vivo transient brain ischemic models. Organotypic hippocampal slice cultures (OHSCs) subjected to oxygen- and glucose-deprivation (OGD) were performed to evaluate in vitro neuroprotective activity of DK extract and nine isolated flavonoid components. MCAO/R mice were employed to elucidate in vivo neuroprotective effects of the flavonoid component that exhibited the most potent neuroprotective effect in OHSCs. DK extract and seven flavonoids [quercetin, isoquercetin, hyperoside, quercetin-3-O-(2″-O-galloyl-β-D-galactopyranoside), kaempferol, astragalin, and kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside) compound (9)] attenuated OGD-induced neuronal cell damage and compound (9) possessed the most potent neuroprotective activity in OHSCs. The MCAO/R mice showed cerebral infarction, massive weight loss, characteristic neurological symptoms, and deterioration of neuronal cells in the brain. Compound (9) and a reference drugs, edaravone, significantly attenuated these physical and neurological impairments. Compound (9) mitigated the blood-brain barrier dysfunction and the change of glutathione and malondialdehyde content in the MCAO mouse brain. Edaravone suppressed the oxidative stress but did not significantly affect the blood-brain barrier permeability. The present results indicated that compound (9) is a flavonoid constituent of DK with a potent neuroprotective activity against transient ischemia-induced brain damage and this action, at least in part, via preservation of blood-brain barrier integrity and suppression of oxidative stress caused by ischemic insult.

Investigating the Mitoprotective Effects of S1P Receptor Modulators Ex Vivo Using a Novel Semi-Automated Live Imaging Set-Up.

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.

Maternal suboptimal selenium intake and low-level lead exposure affect offspring’s microglial immune profile and its reactivity to a subsequent inflammatory hit

Micronutrients such as selenium (Se) are essentials since prenatal life to support brain and cognitive development. Se deficiency, which affects up to 1 billion people worldwide, can interact with common adverse environmental challenges including (Pb), exacerbating their toxic effects. Exploiting our recently validated rat model of maternal Se restriction and developmental low Pb exposure, our aims were to investigate: (i) the early consequences of suboptimal Se intake and low-Pb exposure on neuroinflammation in neonates’ whole brains; (ii) the potential priming effect of suboptimal Se and low-Pb exposure on offspring’s glial reactivity to a further inflammatory hit. To these aims female rats were fed with suboptimal (0.04 mg/kg; Subopt) and optimal (0.15 mg/kg; Opt) Se dietary levels throughout pregnancy and lactation and exposed or not to environmentally relevant Pb dose in drinking water (12.5 µg/mL) since 4 weeks pre-mating. We found an overall higher basal expression of inflammatory markers in neonatal brains, as well as in purified microglia and organotypic hippocampal slice cultures, from the Subopt Se offspring. Subopt/Pb cultures were highly activated than Subopt cultures and showed a higher susceptibility to the inflammatory challenge lipopolysaccharide than cultures from the Opt groups. We demonstrate that even a mild Se deficiency and low-Pb exposure during brain development can influence the neuroinflammatory tone of microglia, exacerbate the toxic effects of Pb and prime microglial reactivity to subsequent inflammatory stimuli. These neuroinflammatory changes may be responsible, at least in part, for adverse neurodevelopmental outcomes.

The cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy

AbstractBackgroundUsing a variety of cell and animal model systems we demonstrate that the cytosolic antibody receptor and E3 ligase TRIM21 plays a critical role in antibody‐mediated protection against tau pathologyMethodExperiments with organotypic hippocampal slice cultures and in vivo studies in mouse models were conducted to study the role of TRIM21 in tau immunotherapy.ResultWe demonstrate that tau:antibody complexes were internalised to the cytosol of neurons, enabling TRIM21 engagement and thereby protecting against seeded aggregation. Crucially, antibody‐mediated protection against tau pathology was lost in mice lacking TRIM21ConclusionThe cytosolic compartment provides a site of immunotherapeutic protection, which can help in the design of antibody‐based therapies in neurodegenerative disease.

C9orf72 dipeptides trigger neuroinflammation via the NLRP3 inflammasome

AbstractBackgroundThe most common cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a repeat expansion in C9orf72. The expansion is translated to produce five toxic dipeptides (DPRs), which aggregate in patient brain. Neuroinflammation is observed in patients, with increased microglial activation and elevated levels of pro‐inflammatory cytokines in CSF. However, the causes of this inflammation are unknown. Here, we investigated whether DPRs activate immune cells and contribute to neuroinflammation.MethodPrimary macrophages or microglia were treated with synthetic C9orf72 dipeptides and levels of pro‐inflammatory cytokines released into the culture media were analysed by ELISA and Western blot. Cytotoxicity was measured by LDH assay. Mouse organotypic hippocampal slice cultures were also treated with DPRs and cytokine release measured.ResultOne of the DPRs, GR, caused secretion of the pro‐inflammatory cytokine, interleukin (IL)‐1β, from macrophages, microglia and in hippocampal slice cultures. GR also caused significant toxicity. IL‐1β secretion was prevented by pre‐treatment with MCC950, a drug which inhibits the NLRP3 inflammasome. The inflammasome is a protein complex which forms in the cytosol of immune cells is response to “danger” stimuli, resulting in cleavage of caspase‐1 and subsequent release of IL‐1β. We confirmed that caspase‐1 was cleaved in macrophages following GR treatment. In addition, knockout of NLRP3 prevented IL‐1β secretion in response to GR. Finally, we showed that several drugs which are clinically approved for use in other conditions rescued GR‐induced NLRP3 activation.ConclusionHere, we demonstrate that C9orf72 dipeptides could directly contribute to neuroinflammation in FTD/ALS via the NLRP3 inflammasome. Furthermore, we demonstrate that this can be inhibited pharmacologically, highlighting NLRP3 as a potential therapeutic target in FTD/ALS.

Immunotherapy against tau pathology in mouse models requires cytosolic Fc receptor TRIM21

AbstractBackgroundThe passive transfer of monoclonal antibodies reduces tau pathology in mouse models and is under evaluation as a potential immunotherapeutic strategy against Alzheimer’s disease and other tauopathies. The mechanisms that drive therapeutic protection of anti‐tau antibodies are not well understood.MethodP301S tau transgenic animals were treated with antibodies against pS422 and analysed for tau pathology by biochemical fractionation. Organotypic hippocampal slice cultures were used as a model of tau seeding and neutralisation. AAV serotypes were used to deliver tau‐selective degraders.ResultGenetic deletion of the cytosolic antibody receptor and E3 ubiquitin ligase TRIM21 rendered tau immunotherapy ineffective in animal models. In organotypic slices, expression of TRIM21 was essential for protection against seeded aggregation of tau. Cytosolic antibody‐tau complexes were visible inside neurons in complex with TRIM21 shortly after application of tau assemblies and antibodies to the cell exterior. Nanobody‐based degraders were developed using TRIM21 and found to protect against tau pathology.ConclusionAnti‐tau antibodies can engage extracellular tau seeds and enter neurons as a complex. Following cytosolic entry, TRIM21 engages the antibodies and mediates neutralisation of tau seeding activity. The results reveal a mechanism that can be optimised for future immunotherapeutics. In this direction, AAV‐delivered degraders reveal a route by which tau aggregates may be selectively removed from the brain.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

The effects of prolyl hydroxylase inhibition during and post, hypoxia, oxygen glucose deprivation and oxidative stress, in isolated rat hippocampal slices

The contributions of hypoxia and oxidative stress to the pathophysiology of acute ischemic stroke are well established and can lead to disruptions in synaptic signaling. Hypoxia and oxidative stress lead to the neurotoxic overproduction of reactive oxygen species (ROS) and the stabilization of hypoxia inducible factors (HIF). Compounds such as prolyl-4-hydroxylase domain enzyme inhibitors (PHDIs) have been shown to have a preconditioning and neuroprotective effect against ischemic insults such as hypoxia, anoxia, oxygen glucose deprivation (OGD) or H2O2. Therefore, this study explored the effects of two PHDIs, JNJ-42041935 (10 µM) and roxadustat (100 µM) on cell viability using organotypic hippocampal slice cultures. We also assessed the effects of these compounds on synaptic transmission during and post hypoxia, OGD and H2O2 application in isolated rat hippocampal slices using field recording electrophysiological techniques and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit trafficking using immunohistochemistry. Our organotypic data demonstrated a protective role for both inhibitors, where slices had significantly less cell death post anoxia and OGD compared to controls. We also report a distinct modulatory role for both JNJ-42041935 and roxadustat on fEPSP slope post hypoxia and OGD but not H2O2. In addition, we report that application of roxadustat impaired long-term potentiation, but only when applied post-hypoxia. This inhibitory effect was not reversed with co-application of the cyclin-dependent kinase 5 (CDK-5) inhibitor, roscovitine (10 µM), suggesting a CDK-5 independent synaptic AMPAR trafficking mechanism. Both hypoxia and OGD induced a reduction in synaptic AMPA GluA2 subunits, the OGD effect being reversed by prior treatment with both JNJ-42041935 and roxadustat. These results suggest an important role for PHDs in synaptic signaling and plasticity during episodes of ischemic stress.

NTS-105 decreased cell death and preserved long-term potentiation in an in vitro model of moderate traumatic brain injury

Traumatic brain injury (TBI) is a major cause of hospitalization and death. To mitigate these human costs, the search for effective drugs to treat TBI continues. In the current study, we evaluated the efficacy of the novel neurosteroid, NTS-105, to reduce post-traumatic pathobiology in an in vitro model of moderate TBI that utilizes an organotypic hippocampal slice culture. NTS-105 inhibited activation of the androgen receptor and the mineralocorticoid receptor, partially activated the progesterone B receptor and was not active at the glucocorticoid receptor. Treatment with NTS-105 starting one hour after injury decreased post-traumatic cell death in a dose-dependent manner, with 10 nM NTS-105 being most effective. Post-traumatic administration of 10 nM NTS-105 also prevented deficits in long-term potentiation (LTP) without adversely affecting neuronal activity in naïve cultures. We propose that the high potency pleiotropic action of NTS-105 beneficial effects at multiple receptors (e.g. androgen, mineralocorticoid and progesterone) provides significant mechanistic advantages over native neurosteroids such as progesterone, which lacked clinical success for the treatment of TBI. Our results suggest that this pleiotropic pharmacology may be a promising strategy for the effective treatment of TBI, and future studies should test its efficacy in pre-clinical animal models of TBI.

Slack K+ channels limit kainic acid-induced seizure severity in mice by modulating neuronal excitability and firing

Mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with terrible epilepsy syndromes that already begin in infancy. Here we report increased severity of acute kainic acid-induced seizures in adult and juvenile Slack knockout mice (Slack−/−) in vivo. Fittingly, we find exacerbation of cell death following kainic acid exposure in organotypic hippocampal slices as well as dissociated hippocampal cultures from Slack−/− in vitro. Furthermore, in cultured Slack−/− neurons, kainic acid-triggered Ca2+ influx and K+ efflux as well as depolarization-induced tetrodotoxin-sensitive inward currents are higher compared to the respective controls. This apparent changes in ion homeostasis could possibly explain altered action potential kinetics of Slack−/− neurons: steeper rise slope, decreased threshold, and duration of afterhyperpolarization, which ultimately lead to higher action potential frequencies during kainic acid application or injection of depolarizing currents. Based on our data, we propose Slack as crucial gatekeeper of neuronal excitability to acutely limit seizure severity.

Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors

BackgroundAdeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. MethodsTwo alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. ResultsCompared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. ConclusionThe substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

In the central nervous system, some specific phosphodiesterase (PDE) isoforms modulate pathways involved in neuronal plasticity. Accumulating evidence suggests that PDE9 may be a promising therapeutic target for neurodegenerative diseases. In the current study, computational techniques were used to identify a nature-inspired PDE9 inhibitor bearing the scaffold of an isoflavone, starting from a database of synthetic small molecules using a ligand-based approach. Furthermore, docking studies supported by molecular dynamics investigations allowed us to evaluate the features of the ligand-target complex. In vitro assays confirmed the computational results, showing that the selected compound inhibits the enzyme in the nanomolar range. Additionally, we evaluated the expression of gene and protein levels of PDE9 in organotypic hippocampal slices, observing an increase following exposure to kainate (KA). Importantly, the PDE9 inhibitor reduced CA3 damage induced by KA in a dose-dependent manner in organotypic hippocampal slices. Taken together, these observations strongly support the potential of the identified nature-inspired PDE9 inhibitor and suggest that such a molecule could represent a promising lead compound to develop novel therapeutic tools against neurological diseases..

Alteration of Neural Network and Hippocampal Slice Activation through Exosomes Derived from 5XFAD Nasal Lavage Fluid.

Exosomes, key mediators of intercellular transmission of pathogenic proteins, such as amyloid-beta and tau, significantly influence the progression and exacerbation of Alzheimer's disease (AD) pathology. Present in a variety of biological fluids, including cerebrospinal fluid, blood, saliva, and nasal lavage fluid (NLF), exosomes underscore their potential as integral mediators of AD pathology. By serving as vehicles for disease-specific molecules, exosomes could unveil valuable insights into disease identification and progression. This study emphasizes the imperative to investigate the impacts of exosomes on neural networks to enhance our comprehension of intracerebral neuronal communication and its implications for neurological disorders like AD. After harvesting exosomes derived from NLF of 5XFAD mice, we utilized a high-density multielectrode array (HD-MEA) system, the novel technology enabling concurrent recordings from thousands of neurons in primary cortical neuron cultures and organotypic hippocampal slices. The ensuing results revealed a surge in neuronal firing rates and disoriented neural connectivity, reflecting the effects provoked by pathological amyloid-beta oligomer treatment. The local field potentials in the exosome-treated hippocampal brain slices also exhibited aberrant rhythmicity, along with an elevated level of current source density. While this research is an initial exploration, it highlights the potential of exosomes in modulating neural networks under AD conditions and endorses the HD-MEA as an efficacious tool for exosome studies.

P10.01.A A PATIENT-DERIVEDEX VIVO ORGANOID MODEL ALLOWS EVALUATION OF TUMOR TREATING FIELDS (TTFIELDS) EFFECTS ON GLIOBLASTOMA, ANAPLASTIC EPENDYMOMA AND OLIGODENDROGLIOMA SAMPLES

Abstract BACKGROUND The use of TTFields, which are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), has shown its clinical efficacy in the treatment of newly diagnosed glioblastoma (GBM) patients at 200 kHz. Nevertheless, more knowledge is required for the prediction of individual tumors’ responsiveness to treatment. Recently, we developed patient-derived three-dimensional GBM tissue culture models to screen patient tumors for TTFields treatment susceptibility. Here, we extended these models to brain tumors beyond GBM, namely anaplastic ependymoma (AE) and oligodendroglioma. We cultured tumor-organoids as microtumors on murine organotypic hippocampal slice cultures (OHSCs) and subjected them to TTFields at 200 kHz. MATERIAL AND METHODS We prepared OHSCs by slicing the brains of mice 5-8 days postpartum to a thickness of 350 µm using a vibratome and culturing them for 14 days. We obtained organoids from freshly resected tumor tissue from three GBM, one AE, and one oligodendroglioma patient, each. The organoids were cultured for 14 days as well and then stained with carboxyfluorescein succinimidyl ester. Next, we placed the organoids onto the OHSCs and administered TTFields at 200 kHz and 1.5 V/cm for 72 h using the inovitro™ laboratory research system. We quantified microtumor growth by fluorescence imaging. RESULTS The three tumor entities, in particular organoids from GBM, AE and oligodendroglioma, grew as microtumors after being placed onto OHSCs. TTFields treatment at 200 kHz led to significantly decreased microtumor size of the three analysed GBM samples as well as the AE sample. However, the oligodendroglioma microtumor size was not significantly reduced. CONCLUSION Based on clinical experience, we expected that TTFields lead to a reduction in microtumor size. However, to our knowledge, we were the first to apply TTFields to patient-derived AE and oligodendroglioma samples. The lack of TTFields-effects on the latter might be due to different inter-patient sensitivity or suboptimal TTFields frequency. Nevertheless, our work is proof of principle that an organoid-based patient-derived 3D tissue culture model could be used to evaluate effects of TTFields on different brain tumor entities.

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a 'before-and-after' experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.