Inhibition of Acute mGluR5-Dependent Depression in Hippocampal CA1 by High-Frequency Magnetic Stimulation.

High-frequency magnetic stimulation (HFMS) applied directly to the hippocampal slice preparation in vitro induces activity-dependent synaptic plasticity and metaplasticity. In addition, changes in synaptic transmission following HFMS involve the activation of N-methyl-D-aspartate and metabotropic glutamate receptors (mGluR). Here, we asked whether a short period of HFMS (5 × 10 delta-burst trains, duration of ~1 min) could alter mGluR5-mediated depression at Schaffer collateral-CA1 synapses in the acute brain slice preparation at 30 min after HFMS. To this end, we obtained field excitatory postsynaptic potential (fEPSP) slopes from Schaffer collateral-CA1 synapses after HFMS or control. First, we demonstrated that activity-dependent plasticity following HFMS depends on the slice orientation towards the magnetic coil indicating specific ion fluxes induced by magnetic fields. Second, we found that the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine reduced the field excitatory postsynaptic potential (fEPSP) slopes in control slices but rather enhanced them in HFMS-treated slices. In contrast, the compound (S)-3,5-dihydroxyphenylglycine acting at both mGluR1 and mGluR5 reduced fEPSP slopes in both control and HFMS-treated slices. Importantly, the mGluR-dependent effects were independent from the slice-to-coil orientation indicating that asynchronous glutamate release could play a role. We conclude that a short period of HFMS inhibits subsequently evoked mGluR5-dependent depression at Schaffer collateral-CA1 synapses. This could be relevant for repetitive transcranial magnetic stimulation in psychiatric disorders such as major depression.

A glutamatergic biomarker panel enables differentiating Grade 4 gliomas/astrocytomas from brain metastases.

Background: The differentiation of high-grade glioma and brain tumors of an extracranial origin is eminent for the decision on subsequent treatment regimens. While in high-grade glioma, a surgical resection of the tumor mass is a fundamental part of current standard regimens, in brain metastasis, the burden of the primary tumor must be considered. However, without a cancer history, the differentiation remains challenging in the imaging. Hence, biopsies are common that may help to identify the tumor origin. An additional tool to support the differentiation may be of great help. For this purpose, we aimed to identify a biomarker panel based on the expression analysis of a small sample of tissue to support the pathological analysis of surgery resection specimens. Given that an aberrant glutamate signaling was identified to drive glioblastoma progression, we focused on glutamate receptors and key players of glutamate homeostasis. Methods: Based on surgically resected samples from 55 brain tumors, the expression of ionotropic and metabotropic glutamate receptors and key players of glutamate homeostasis were analyzed by RT-PCR. Subsequently, a receiver operating characteristic (ROC) analysis was performed to identify genes whose expression levels may be associated with either glioblastoma or brain metastasis. Results: Out of a total of 29 glutamatergic genes analyzed, nine genes presented a significantly different expression level between high-grade gliomas and brain metastases. Of those, seven were identified as potential biomarker candidates including genes encoding for AMPA receptors GRIA1, GRIA2, kainate receptors GRIK1 and GRIK4, metabotropic receptor GRM3, transaminase BCAT1 and the glutamine synthetase (encoded by GLUL). Overall, the biomarker panel achieved an accuracy of 88% (95% CI: 87.1, 90.8) in predicting the tumor entity. Gene expression data, however, could not discriminate between patients with seizures from those without. Conclusion: We have identified a panel of seven genes whose expression may serve as a biomarker panel to discriminate glioblastomas and brain metastases at the molecular level. After further validation, our biomarker signatures could be of great use in the decision making on subsequent treatment regimens after diagnosis.

Persistent Kv7.2/7.3 downregulation in the rat pilocarpine model of mesial temporal lobe epilepsy.

Mutations within the Kv7.2 and Kv7.3 genes are well described causes for genetic childhood epilepsies. Knowledge on these channels in acquired focal epilepsy, especially in mesial temporal lobe epilepsy (mTLE), however, is scarce. Here, we used the rat pilocarpine model of drug-resistant mTLE to elucidate both expression and function by quantitative polymerase-chain reaction, immunohistochemistry, and electrophysiology, respectively. We found transcriptional downregulation of Kv7.2 and Kv7.3 as well as reduced Kv7.2 expression in epileptic CA1. Consequences were altered synaptic transmission, hyperexcitability which consisted of epileptiform afterpotentials, and increased susceptibility to acute GABAergic disinhibition. Importantly, blocking Kv7 channels with XE991 increased hyperexcitability in control tissue, but not in chronically epileptic tissue suggesting that the Kv7 deficit had precluded XE991 effects in this tissue. Conversely, XE991 resulted in comparable reduction of the paired-pulse ratio in both experimental groups implying preserved presynaptic Kv7.2 function of Schaffer collateral terminals. Consistent with Kv7.2/7.3 downregulation, the Kv7.3 channel opener ß-hydroxybutyrate failed to mitigate hyperexcitability. Our findings demonstrate that compromised Kv7 function is not only relevant in genetic epilepsy, but also in acquired focal epilepsy. Moreover, they help explain reduced anti-seizure efficacy of Kv7 channel openers in drug-resistant epilepsy.

Age-dependent effects of the ß3 adrenoceptor agonist CL316,243 on human and rat detrusor muscle strips.

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. ß3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 µM) in the presence of the ß3 adrenoceptor agonist CL316,243 (from 0.1 to 10 µM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by ß3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen's d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC50. Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved ß3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human ß3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to ß3 agents.

Direct-Current Electrical Field Stimulation of Patient-Derived Colorectal Cancer Cells.

Several cues for a directional migration of colorectal cancer cells were identified as being crucial in tumor progression. However, galvanotaxis, the directional migration in direct-current electrical fields, has not been investigated so far. Therefore, we asked whether direct-current electrical fields could be used to mobilize colorectal cancer cells along field vectors. For this purpose, five patient-derived low-passage cell lines were exposed to field strengths of 150-250 V/m in vitro, and migration along the field vectors was investigated. To further study the role of voltage-gated calcium channels on galvanotaxis and intracellular signaling pathways that are associated with migration of colorectal cancer cells, the cultures were exposed to selective inhibitors. In three out of five colorectal cancer cell lines, we found a preferred cathodal migration. The cellular integrity of the cells was not impaired by exposure of the cells to the selected field strengths. Galvanotaxis was sensitive to inhibition of voltage-gated calcium channels. Furthermore, signaling pathways such as AKT and MEK, but not STAT3, were also found to contribute to galvanotaxis in our in vitro model system. Overall, we identify electrical fields as an important contributor to the directional migration of colorectal cancer cells.

Functional changes in neuronal circuits due to antibody-driven autoimmune response.

Autoimmune-mediated encephalitis syndromes are increasingly being recognized as important clinical entities. They need to be thought of as differential diagnosis in any patient presenting with fast-onset psychosis or psychiatric problems, memory deficits or other cognitive problems, including aphasias, as well as seizures or motor automatisms, but also rigidity, paresis, ataxia or dystonic / parkinsonian symptoms. Diagnosis including imaging and CSF search for antibodies needs to be fast, as progression of these inflammatory processes is often causing scarring of brain tissue, with hypergliosis and atrophy. As these symptoms show, the autoantibodies present in these cases appear to act within the CNS. Several of such antibodies have by now been identified such as IgG directed against NMDA-receptors, AMPA receptors, GABAA and GABAB receptors, and voltage gated potassium channels and proteins of the potassium channel complex (i.e. LGI1 and CASPR2). These are neuropil / surface antigens where antibody interaction can well be envisaged to cause dysfunction of the target protein, including internalization. Others, such as antibodies directed against GAD65 (an intracellular enzyme responsible for GABA-synthesis from glutamate), are discussed to constitute epiphenomena, but not causal agents in disease progression. This review will focus on the current knowledge of antibody interaction mechanisms, especially discussing cellular excitability changes and synaptic interactions in hippocampal and other brain networks. One challenge in this context is to find viable hypotheses for the emergence of both, hyperexcitability and seizures, and presumably reduced synaptic plasticity and underlying cognitive dysfunction.

[Reported and measured olfactory and gustatory findings in a long-COVID cohort]. / Berichtetes und gemessenes Riech- und Schmeckvermögen in einer Long-COVID-Kohorte.

INTRODUCTION: Disorders of the sense of smell and taste are often complained as a long-COVID symptom. In a special long-COVID consultation (ENT-LCS), we wanted to figure out how large the proportion of pathological olfactory or gustatory diagnoses actually is in this cohort. METHODS: 48 adult patients who visited the ENT-LCS because of their own suffering were asked about their history, rated their ability to smell (SER) and taste (SES) with school grades and completed the extended Sniffin' Sticks test and the 3-drop test as a taste test. Diagnoses were made from the SDI smell score and the total taste score using normative values. Correlations were calculated between the measured scores and the self-assessment and between SER and SES. RESULTS: Pathological chemosensory diagnoses were present in 90%. Pathological olfactory diagnoses were twice as common as gustatory ones. No pathological diagnosis could be verified in 10% of the cohort. Anosmia-ageusia syndrome was diagnosed in six patients. SER correlated strongly with SDI. SES correlated moderately with SScore-total. SER correlated strongly with SES. CONCLUSIONS: There is a risk of smell-taste confusion when interpreting reported chemosensory symptoms. This is another reason why reference is made to the importance of quantifying the chemical senses with validated tests.

GluN2B inhibition rescues impaired potentiation and epileptogenicity at associational-commissural CA3 synapses in a model of anti-NMDAR encephalitis.

Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune epilepsy associated with memory deficits. Research has demonstrated that anti-NMDAR inhibit long-term potentiation, and, at the same time, lead to disinhibition in the form of epileptiform afterpotentials in the potentiated state. While both effects may give rise to the key symptoms of the disease, the molecular basis of being simultaneously inhibitory and disinhibitory is difficult to explain. Here, we explored a possible involvement of the GluN2B subunit. To this aim, we injected cerebrospinal fluid from anti-NMDAR encephalitis patients into the rat hippocampus and prepared brain slices for in vitro field potential recordings. Associational-commissural-fiber-CA3 synapses from anti-NMDAR-treated animals showed increased field potential amplitudes with concomitantly enhanced paired-pulse ratios as compared to control tissue. GluN2B inhibition by Ro25-6981 mimicked these effects in controls but had no effect in anti-NMDAR tissues indicating a presynaptic and occluding effect of anti-NMDAR. We then induced potentiation of associational-commissural-fiber-CA3 synapses, and confirmed that slices from anti-NMDAR-treated animals showed reduced potentiation and pronounced epileptiform afterpotentials. Intriguingly, both effects were absent when Ro25-6981 was added in vitro before inducing potentiation. These results indicate that GluN2B-containing NMDARs, partially expressed presynaptically, show differential sensitivity to anti-NMDAR, and that altered GluN2B function is particularly apparent in the potentiated state rather than under baseline conditions. Since GluN2B inhibition rescued the effects of anti-NMDAR in the potentiated state, this opens the possibility that at least a subgroup of patients could benefit from a GluN2B antagonist.

Studies on Biological and Molecular Effects of Small-Molecule Kinase Inhibitors on Human Glioblastoma Cells and Organotypic Brain Slices.

Glioblastoma is the most common and aggressive primary brain tumor. Multiple genetic and epigenetic alterations in several major signaling pathways-including the phosphoinositide 3-kinases (PI3K)/AKT/mTOR and the Raf/MEK/ERK pathway-could be found. We therefore aimed to investigate the biological and molecular effects of small-molecule kinase inhibitors that may interfere with those pathways. For this purpose, patient-derived glioblastoma cells were challenged with dactolisib, ipatasertib, MK-2206, regorafenib, or trametinib. To determine the effects of the small-molecule kinase inhibitors, assays of cell proliferation and apoptosis and immunoblot analyses were performed. To further investigate the effects of ipatasertib on organotypic brain slices harboring glioblastoma cells, the tumor growth was estimated. In addition, the network activity in brain slices was assessed by electrophysiological field potential recordings. Multi-kinase inhibitor regorafenib and both MK-2206 and dactolisib were very effective in all preclinical tumor models, while with respect to trametinib, two cell lines were found to be highly resistant. Only in HROG05 cells, ipatasertib showed anti-tumoral effects in vitro and in organotypic brain slices. Additionally, ipatasertib diminished synchronous network activity in organotypic brain slices. Overall, our data suggest that ipatasertib was only effective in selected tumor models, while especially regorafenib and MK-2206 presented a uniform response pattern.

Modification of the Langendorff system of the isolated beating heart for experimental radiotherapy at a synchrotron: 4000†Gy in a heart beat.

Microbeam radiotherapy could help to cure malignant tumours which are currently still considered therapy-resistant. With an irradiation target in the thoracic cavity, the heart would be one of the most important organs at risk. To assess the acute adverse effects of microbeam irradiation in the heart, a powerful ex vivo tool was created by combining the Langendorff model of the isolated beating mammalian heart with X-Tream dosimetry. In a first pilot experiment conducted at the Biomedical and Imaging Beamline of the Australian Synchrotron, the system was tested at a microbeam peak dose approximately ten times higher than the anticipated future microbeam irradiation treatment doses. The entire heart was irradiated with a dose of 4000 Gy at a dose rate of >6000 Gy s-1, using an array of 50 µm-wide microbeams spaced at a centre-to-centre distance of 400 µm. Although temporary arrhythmias were seen, they reverted spontaneously to a stable rhythm and no cardiac arrest occurred. This amazing preservation of cardiac function is promising for future therapeutic approaches.

Microbeam Irradiation of the Beating Rodent Heart: An Ex Vivo Study of Acute and Subacute Effects on Cardiac Function.

PURPOSE: Microbeam radiation therapy (MRT) has shown several advantages compared with conventional broad-beam radiation therapy in small animal models, including a better preservation of normal tissue function and improved drug delivery based on a rapidly increased vascular permeability in the target region. Normal tissue tolerance is the limiting factor in clinical radiation therapy. Knowledge of the normal tissue tolerance of organs at risk is therefore a prerequisite in evaluating any new radiation therapy approach. With an irradiation target in the thoracic cavity, the heart would be the most important organ at risk. METHODS AND MATERIALS: We used the ex vivo beating rodent heart in the Langendorff perfusion system at the synchrotron to administer microbeam irradiation (MBI) with a peak dose of 40 or 400 Gy. By continuously recording the electrocardiogram, the left ventricular pressure, and the aortic pressure before, during and after MBI, we were able to assess acute and subacute effects of MBI on electrophysiological and mechanical cardiac function. In addition, we analyzed histologic and ultrastructural sequelae caused by MBI. RESULTS: There were no significant changes in heart rate, heart rate variability, systolic increase of left ventricular pressure or aortic pressure. Moreover, the changes of heart rate, left ventricular pressure and aortic pressure by adding 10-5 mol/L norepinephrine to the perfusate, were also not significant between MBI and sham experiments. However, the rate-pressure product as a surrogate marker for maximum workload after MBI was significantly lower compared with sham-irradiated controls. On the structural level, no severe membranous, sarcomeric, mitochondrial or nuclear changes caused by MBI were detected by desmin immunohistochemistry and electron microscopy. CONCLUSIONS: With respect to acute and subacute toxicity, an MBI peak dose up to 400 Gy did not result in severe changes in cardiac electrophysiology or mechanics.

Galvanotactic Migration of Glioblastoma and Brain Metastases Cells.

Galvanotaxis, the migration along direct current electrical fields, may contribute to the invasion of brain cancer cells in the tumor-surrounding tissue. We hypothesized that pharmacological perturbation of the epidermal growth factor (EGF) receptor and downstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevent galvanotactic migration. In our study, patient-derived glioblastoma and brain metastases cells were exposed to direct current electrical field conditions. Velocity and direction of migration were estimated. To determine the effects of EGF receptor antagonist afatinib and AKT inhibitor capivasertib, assays of cell proliferation, apoptosis and immunoblot analyses were performed. Both inhibitors attenuated cell proliferation in a dose-dependent manner and induced apoptosis. We found that most of the glioblastoma cells migrated preferentially in an anodal direction, while brain metastases cells were unaffected by direct current stimulations. Afatinib presented only a mild attenuation of galvanotaxis. In contrast, capivasertib abolished the migration of glioblastoma cells without genetic alterations in the PI3K/AKT pathway, but not in cells harboring PTEN mutation. In these cells, an increase in the activation of ERK1/2 may in part substitute the inhibition of the AKT pathway. Overall, our data demonstrate that glioblastoma cells migrate in the electrical field and the PI3K/AKT pathway was found to be highly involved in galvanotaxis.

High prevalence of long-term olfactory dysfunction confirmed by olfactory testing after a community COVID-19 outbreak.

PURPOSE: The prevalence of long-term olfactory and gustatory dysfunction in participants suffering from sudden chemosensory loss due to coronavirus disease 2019 (COVID-19) is unknown. Furthermore, evaluations of the reliability of participants' self-reporting of olfactory function (SOF) and gustatory function (SGF) using extended objective psychophysical testing are missing. METHODS: In this population-based cohort study in a PCR-tested community in Thuringia, Germany, olfactory function was extensively examined 4 months after a COVID-19 outbreak using the "Sniffin Sticks" test battery to determine the TDIa score, i.e., the sum of results obtained for threshold, discrimination, and identification scores averaged for both nasal sides. Gustatory function was assessed using the three-drop test resulting in the gustatory composite score (CSg). The data were compared with SOF and SGF. RESULTS: Of 43 adult convalescents (median age: 68 years; 58% female) after SARS-CoV­2 infection, 18 participants (42%) had olfactory complaints due to SOF, one participant (2%) complained of taste disturbance due to SGF. The TDIa was 22.0 ± 5.9. Normosmia, hyposmia, and anosmia were seen in 17, 18, and eight participants, respectively. TDIa correlated with SOF (rs = -0.434, p = 0.004); CSg was 23.5 ± 2.7. Normogeusia and hypogeusia were objectified in 39 and four participants, respectively. The prevalence of long-term olfactory dysfunction and gustatory dysfunction in the study group was 60.5 and 9.3%, respectively. CONCLUSION: The SOF was reliable, especially for participants who felt a sudden chemosensory dysfunction during the outbreak. At 4 months after SARS-CoV­2 infection, a high proportion of participants were dysosmic, whereas nearly all of them had normal taste function.

Effects of Microbeam Irradiation on Rodent Esophageal Smooth Muscle Contraction.

BACKGROUND: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. METHODS: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy. RESULTS: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. CONCLUSION: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.

Glutamatergic Mechanisms in Glioblastoma and Tumor-Associated Epilepsy.

The progression of glioblastomas is associated with a variety of neurological impairments, such as tumor-related epileptic seizures. Seizures are not only a common comorbidity of glioblastoma but often an initial clinical symptom of this cancer entity. Both, glioblastoma and tumor-associated epilepsy are closely linked to one another through several pathophysiological mechanisms, with the neurotransmitter glutamate playing a key role. Glutamate interacts with its ionotropic and metabotropic receptors to promote both tumor progression and excitotoxicity. In this review, based on its physiological functions, our current understanding of glutamate receptors and glutamatergic signaling will be discussed in detail. Furthermore, preclinical models to study glutamatergic interactions between glioma cells and the tumor-surrounding microenvironment will be presented. Finally, current studies addressing glutamate receptors in glioma and tumor-related epilepsy will be highlighted and future approaches to interfere with the glutamatergic network are discussed.

Kv7 and Kir6 Channels Shape the Slow AHP in Mouse Dentate Gyrus Granule Cells and Control Burst-like Firing Behavior.

The slow afterhyperpolarizing potential (sAHP) can silence a neuron for hundreds of milliseconds. Thereby, the sAHP determines the discharge behavior of many types of neurons. In dentate granule cells (DGCs), serving as a filter into the hippocampal network, mostly tonic or adapting discharge properties have been described. As under standard whole-cell recording conditions the sAHP is inhibited, we reevaluated the intrinsic functional phenotype of DGCs and the conductances underlying the sAHP, using gramicidine-perforated patch-clamp technique. We found that in 97/113 (86%) of the DGCs, a burst of action potentials (APs) to excitation ended by a large sAHP, despite continued depolarization. This result suggests that burst-like firing is the default functional phenotype of DGCs and that sAHPs are important for it. Indeed, burst-like firing DGCs showed a significantly higher sAHP-current (IsAHP) amplitude compared to spike-frequency adapting cells (16/113 = 14%). The IsAHP was mediated by Kv7 and Kir6 channels by pharmacological inhibition using XE991 and tolbutamide, although heterogeneously among DGCs. The percent inhibition of IsAHP by these compounds also correlated with the AP number and AP burst length. Application of 100 µM nickel after XE991 and tolbutamide detected a third conductance contributing to burst-like firing and the sAHP, most likely mediated by T-type calcium channels. Lastly, medial perforant path-dentate gyrus long-term potentiation was amplified by XE991 and tolbutamide. In conclusion, the sAHP shapes intrinsic burst-like firing which, under physiological circumstances, could be controlled via cholinergic afferents and ATP metabolism.

Neuronal Hyperexcitability in APPSWE/PS1dE9 Mouse Models of Alzheimer's Disease.

Transgenic mouse models serve a better understanding of Alzheimer's disease (AD) pathogenesis and its consequences on neuronal function. Well-known and broadly used AD models are APPswe/PS1dE9 mice, which are able to reproduce features of amyloid-ß (Aß) plaque formations as well as neuronal dysfunction as reflected in electrophysiological recordings of neuronal hyperexcitability. The most prominent findings include abnormal synaptic function and synaptic reorganization as well as changes in membrane threshold and spontaneous neuronal firing activities leading to generalized excitation-inhibition imbalances in larger neuronal circuits and networks. Importantly, these findings in APPswe/PS1dE9 mice are at least partly consistent with results of electrophysiological studies in humans with sporadic AD. This underscores the potential to transfer mechanistic insights into amyloid related neuronal dysfunction from animal models to humans. This is of high relevance for targeted downstream interventions into neuronal hyperexcitability, for example based on repurposing of existing antiepileptic drugs, as well as the use of combinations of imaging and electrophysiological readouts to monitor effects of upstream interventions into amyloid build-up and processing on neuronal function in animal models and human studies. This article gives an overview on the pathogenic and methodological basis for recording of neuronal hyperexcitability in AD mouse models and on key findings in APPswe/PS1dE9 mice. We point at several instances to the translational perspective into clinical intervention and observation studies in humans. We particularly focus on bi-directional relations between hyperexcitability and cerebral amyloidosis, including build-up as well as clearance of amyloid, possibly related to sleep and so called glymphatic system function.

Correlation between Kir4.1 expression and barium-sensitive currents in rat and human glioma cell lines.

Gliomas are the most common primary brain tumors and often become apparent through symptomatic epileptic seizures. Glial cells express the inwardly rectifying K+ channel Kir4.1 playing a major role in K+ buffering, and are presumably involved in facilitating epileptic hyperexcitability. We therefore aimed to investigate the molecular and functional expression of Kir4.1 channels in cultured rat and human glioma cells. Quantitative PCR showed reduced expression of Kir4.1 in rat C6 and F98 cells as compared to control. In human U-87MG cells and in patient-derived low-passage glioblastoma cultures, Kir4.1 expression was also reduced as compared to autopsy controls. Testing Kir4.1 function using whole-cell patch-clamp experiments on rat C6 and two human low-passage glioblastoma cell lines (HROG38 and HROG05), we found a significantly depolarized resting membrane potential (RMP) in HROG05 (-29 ± 2 mV, n = 11) compared to C6 (-71 ± 1 mV, n = 12, P < 0.05) and HROG38 (-60 ± 2 mV, n = 12, P < 0.05). Sustained K+ inward or outward currents were sensitive to Ba2+ added to the bath solution in HROG38 and C6 cells, but not in HROG05 cells, consistent with RMP depolarization. While immunocytochemistry confirmed Kir4.1 in all three cell lines including HROG05, we found that aquaporin-4 and Kir5.1 were also significantly reduced suggesting that the Ba2+-sensitive K+ current is generally impaired in glioma tissue. In summary, we demonstrated that glioma cells differentially express functional inwardly rectifying K+ channels suggesting that impaired K+ buffering in cells lacking functional Ba2+-sensitive K+ currents may be a risk factor for increased excitability and thereby contribute to the differential epileptogenicity of gliomas.

Perampanel Add-on to Standard Radiochemotherapy in vivo Promotes Neuroprotection in a Rodent F98 Glioma Model.

An abnormal glutamate signaling of glioblastoma may contribute to both tumor progression and the generation of glioma-associated epileptic seizures. We hypothesized that the AMPA receptor antagonist perampanel (PER) could attenuate tumor growth and epileptic events. F98 glioma cells, grown orthotopically in Fischer rats, were employed as a model of glioma to investigate the therapeutic efficiency of PER (15 mg/kg) as adjuvant to standard radiochemotherapy (RCT). The epileptiform phenotype was investigated by video-EEG analysis and field potential recordings. Effects on glioma progression were estimated by tumor size quantification, survival analysis and immunohistological staining. Our data revealed that orthotopically-growing F98 glioma promote an epileptiform phenotype in rats. RCT reduced the tumor size and prolonged the survival of the animals. The adjuvant administration of PER had no effect on tumor progression. The tumor-associated epileptic events were abolished by PER application or RCT respectively, to initial baseline levels. Remarkably, PER preserved the glutamatergic network activity on healthy peritumoral tissue in RCT-treated animals. F98 tumors are not only a robust model to investigate glioma progression, but also a viable model to simulate a glioma-associated epileptiform phenotype. Furthermore, our data indicate that PER acts as a potent anticonvulsant and may protect the tumor-surrounding tissue as adjuvant to RCT, but failed to attenuate tumor growth or promote animal survival.

Stereotactically Injected Kv1.2 and CASPR2 Antisera Cause Differential Effects on CA1 Synaptic and Cellular Excitability, but Both Enhance the Vulnerability to Pro-epileptic Conditions.

PURPOSE: We present a case of voltage-gated potassium channel (VGKC) complex antibody-positive limbic encephalitis (LE) harboring autoantibodies against Kv1.2. Since the patient responded well to immunotherapy, the autoantibodies were regarded as pathogenic. We aimed to characterize the pathophysiological role of this antibody in comparison to an antibody against the VGKC-associated protein contactin-associated protein-2 (CASPR2). METHODS: Stereotactic injection of patient sera (anti-Kv1.2-associated LE or anti-CASPR2 encephalopathy) and a control subject was performed into the hippocampus of the anesthetized rat in vivo, and hippocampal slices were prepared for electrophysiological purposes. Using extra- and intracellular techniques, synaptic transmission, long-term potentiation (LTP) and vulnerability to pro-epileptic conditions were analyzed. RESULTS: We observed that the slope of the field excitatory postsynaptic potential (fEPSP) was significantly increased at Schaffer collateral-CA1 synapses in anti-Kv1.2-treated and anti-CASPR2-treated rats, but not at medial perforant path-dentate gyrus synapses. The increase of the fEPSP slope in CA1 was accompanied by a decrease of the paired-pulse ratio in anti-Kv1.2, but not in anti-CASPR2 tissue, indicating presynaptic site of anti-Kv1.2. In addition, anti-Kv1.2 tissue showed enhanced LTP in CA1, but dentate gyrus LTP remained unaltered. Importantly, LTP in slices from anti-CASPR2-treated animals did not differ from control values. Intracellular recordings from CA1 neurons revealed that the resting membrane potential and a single action potential were not different between anti-Kv1.2 and control tissue. However, when the depolarization was prolonged, the number of action potentials elicited was reduced in anti-Kv1.2-treated tissue compared to both control and anti-CASPR2 tissue. In contrast, polyspike discharges induced by removal of Mg2+ occurred earlier and more frequently in both patient sera compared to control. CONCLUSION: Patient serum containing anti-Kv1.2 facilitates presynaptic transmitter release as well as postsynaptic depolarization at the Schaffer-collateral-CA1 synapse, but not in the dentate gyrus. As a consequence, both synaptic transmission and LTP in CA1 are facilitated and action potential firing is altered. In contrast, anti-CASPR2 leads to increased postsynaptic potentials, but without changing LTP or firing properties suggesting that anti-Kv1.2 and anti-CASPR2 differ in their cellular effects. Both patient sera alter susceptibility to epileptic conditions, but presumably by different mechanisms.