Development of a rat model for glioma-related epilepsy

Seizures are common in patients with high-grade gliomas (30–60%) and approximately 15–30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopaty

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer’s disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer’s disease

Extracellular recordings of neuronal activity using multi-electrode arrays: application to acute and organotypic hippocampal slices

Multi-electrode arrays, as efficient in vitro test-platforms to record electrical activity from excitable cells, have many advantages over conventional methods using glass microelectrodes or patch pipettes. MEAs consist of 60 to thousands electrodes that are individually and often simultaneously addressable for stimulation or recording, therefore offer excellent spatial resolution. MEAs are also particularly valuable for long-term electrophysiological studies since the planar or micronail-shaped extracellular electrodes can get close to a cell without damaging. Despite of these advantages, the use of MEAs is not yet standardized and only a few optimized protocols and applications have emerged in the field of neuroscience. This PhD project therefore aimed to develop and optimize protocols that allow us to use commercially available MEAs as well as advanced CMOS-based MEAs to study neuronal transmission and synaptic plasticity in brain tissues. In the first part of this thesis, using commercially available MEAs, we recorded synaptic transmission and plasticity simultaneously in different hippocampal subregions without cross-talk interference. We investigated early changes in synaptic function and plasticity in the CA1 and CA3 region of young APP.V717I, Tau.P301L and biAT transgenic mice as models for amyloid and/or tau-pathology in Alzheimer s disease. Our findings demonstrate that long before evidence of plaque or tangle formation important subregional synaptic changes occur in the hippocampus. The MEA platform was further used to examine direct effects of human mutant tau expression on the hippocampal temporoammonic transmission in adeno-associated viral vector (AAV)-Tau.P301L injected mice. Independent recording and stimulation of the temporoammonic pathway were successfully performed. The results show that in AAV-Tau.P301L injected mice short-term plasticity in the temporoammonic pathway is significantly impaired while long-term plasticity is not affected. Finally, MEA recordings were used to characterize an Alzheimer s disease animal model generated by microRNA 29a/b-1 cluster knock-out. The results of this study showed that in microRNA 29a/b-1 knock-out mice both basal and short-term synaptic transmissions are impaired while long-term potentiation is unaffected. In the second part of this thesis, we focused on developing and optimizing organotypic slice cultures integrated with complementary metal oxide semiconductor (CMOS)-based MEA surfaces to overcome limitations of acute slice applications. An automated slice tilter was developed to maintain brain slices at the interface between medium and humidified atmosphere at long-term. Slices cultured for 2 weeks showed good general viability and retained the intact hippocampal cytoarchitecture. We also found that CMOS-based high-density MEAs featuring thousands of micronail electrodes significantly enhanced initial slice adhesion therefore improved general viability compared to flat MEA surfaces. In conclusion, this work demonstrates that MEAs provide a highly stable system for the long-term monitoring of spontaneous or evoked neuronal activity in acute or organotypic brain slices and considerably improve read-out or low-throughput of conventional electrophysiological methods. In addition, the integration of organotypic brain slices with CMOS high-density MEAs is an attractive biosensor system that has the potential to address, manipulate and record electrical activity of each individual cell within a neuronal network.nrpages: 171status: publishe

Synaptic dysfunction in hippocampus of transgenic mouse models of Alzheimer's disease: A multi-electrode array study

APP.V717I and Tau.P301L transgenic mice develop Alzheimer's disease pathology comprising important aspects of human disease including increased levels of amyloid peptides, cognitive and motor impairment, amyloid plaques and neurofibrillary tangles. The combined model, APP.V717I×Tau.P301L bigenic mice (biAT mice) exhibit aggravated amyloid and tau pathology with severe cognitive and behavioral defects. In the present study, we investigated early changes in synaptic function in the CA1 and CA3 regions of acute hippocampal slices of young APP.V717I, Tau.P301L and biAT transgenic animals. We have used planar multi-electrode arrays (MEA) and improved methods for simultaneous multi-site recordings from two hippocampal sub-regions. In the CA1 region, long-term potentiation (LTP) was severely impaired in all transgenic animals when compared with age-matched wild-type controls, while basal synaptic transmission and paired-pulse facilitation were minimally affected. In the CA3 region, LTP was normal in Tau.P301L and APP.V717I but clearly impaired in biAT mice. Surprisingly, frequency facilitation in CA3 was significantly enhanced in Tau.P301L mice, while not affected in APP.V717I mice and depressed in biAT mice. The findings demonstrate important synaptic changes that differ considerably in the hippocampal sub-regions already at young age, well before the typical amyloid or tau pathology is evident.status: publishe

Parameters of dendritic spines in CA1 SLM of wild-type FvB and Tau.P301L mice.

<p>Parameters of dendritic spines in CA1 SLM of wild-type FvB and Tau.P301L mice.</p

Timeline of phospho-Tau in hippocampal formation of Tau.P301L and biGT mice.

<p>IHC for pT231-Tau (AT180) and pS199–202 (AT8) in the sub-regions of the hippocampal formation of Tau.P301L and biGT mice at 3 months of age and at terminal stage (see text for details). Red asterisk marks CA1 pyramidal layer degeneration. Images are representative for the median levels of AT180 and AT8 staining (n = 6–8 mice per age group). Denoted are layers I, II and III of the entorhinal cortex and CA1 hippocampal sub-regions stratum radiatum (SR) and stratum lacunosum moleculare (SLM).</p

Timeline of phospho-Tau in the CA1 SLM of Tau.P301L and biGT mice.

<p>IHC for AT180 (A) and AT8 (B) staining in CA1 SLM region of Tau.P301L and biGT mice at ages 3, 6 and 9 months and terminal. Data are represented as median intensity score for immunoreactivity with AT180 (A) and AT8 (B) in CA1 SLM. Grey symbols denote mice with clasping and black symbols denote terminal mice (see text for details)(n = 6–8 mice per age group). CA1 hippocampal sub-regions stratum radiatum (SR) and stratum lacunosum molecular (SLM) are marked.</p

Myelin defects in SLM of Tau.P301L mice.

<p>A. Representative images of the ultrastructure of myelinated axons in wild-type, Tau.P301L and biGT mice, with myelin defects indicated by the symbols explained in the caption. B. G-ratio distribution of TA axons in SLM in wild-type FvB, Tau.P301L and biGT mice (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087605#pone-0087605-t002" target="_blank">Table 2</a>). C. Quantitation of axons harboring morphological defects (left panel) and of axonal length with disrupted myelin sheaths (right panel) in TA axons of SLM in the three genotypes. Data are mean±SEM, statistically analyzed by one-way ANOVA followed by Bonferroni Multiple Comparison Test post hoc test; *p<0.05, **p<0.01, ***p<0.001.</p