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Traumatic brain injury (TBI) causes 10–20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. Previous studies revealed TBI-induced alterations in blood microRNA (miRNA) levels, and patients with epilepsy exhibit dysregulation of blood miRNAs. We hypothesized that acutely altered plasma miRNAs could serve as prognostic biomarkers for brain damage severity and the development of PTE. To investigate this, epileptogenesis was induced in adult male Sprague Dawley rats by lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least one unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Cortical pathology was analyzed by magnetic resonance imaging on day 2 (D2), D7, and D21, and by histology 6 months post-TBI. Small RNA sequencing was performed from tail-vein plasma samples on D2 and D9 after TBI (n = 16, 7 with and 9 without epilepsy) or sham operation (n = 4). The most promising miRNA biomarker candidates were validated by droplet digital polymerase chain reaction in a validation cohort of 115 rats (8 naïve, 17 sham, and 90 TBI rats [21 with epilepsy]). These included 7 brain-enriched plasma miRNAs (miR-434-3p, miR-9a-3p, miR-136-3p, miR-323-3p, miR-124-3p, miR-212-3p, and miR-132-3p) that were upregulated on D2 post-TBI (p < 0.001 for all compared with naïve rats). The acute post-TBI plasma miRNA profile did not predict the subsequent development of PTE or PTE severity. Plasma miRNA levels, however, predicted the cortical pathology severity on D2 (Spearman ρ = 0.345–0.582, p < 0.001), D9 (ρ = 0.287–0.522, p < 0.001–0.01), D21 (ρ = 0.269–0.581, p < 0.001–0.05) and at 6 months post-TBI (ρ = 0.230–0.433, p < 0.001–0.05). We found that the levels of 6 of 7 miRNAs also reflected mild brain injury caused by the craniotomy during sham operation (ROC AUC 0.76–0.96, p < 0.001–0.05). In conclusion, our findings revealed that increased levels of neuronally enriched miRNAs in the blood circulation after TBI reflect the extent of cortical injury in the brain but do not predict PTE development.

Background The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70–80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes – cell-adhesion molecule contactin-3. Methods Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1–48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0–44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0–3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro. Results CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = − 0.5, p CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p Conclusions Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.

Recurrent processing is a crucial feature in human visual processing supporting perceptual grouping, figure-ground segmentation, and recognition under challenging conditions. There is a clear need to incorporate recurrent processing in deep convolutional neural networks (DCNNs) but the computations underlying recurrent processing remain unclear. In this paper, we tested a form of recurrence in deep residual networks (ResNets) to capture recurrent processing signals in the human brain. Though ResNets are feedforward networks, they approximate an excitatory additive form of recurrence. Essentially, this form of recurrence consists of repeating excitatory activations in response to a static stimulus. Here, we used ResNets of varying depths (reflecting varying levels of recurrent processing) to explain electroencephalography (EEG) activity within a visual masking paradigm. Sixty-two humans and fifty artificial agents (10 ResNet models of depths - 4, 6, 10, 18 and 34) completed an object categorization task. We show that deeper networks (ResNet-10, 18 and 34) explained more variance in brain activity compared to shallower networks (ResNet-4 and 6). Furthermore, all ResNets captured differences in brain activity between unmasked and masked trials, with differences starting at ∼98ms (from stimulus onset). These early differences indicated that EEG activity reflected ‘pure’ feedforward signals only briefly (up to ∼98ms). After ∼98ms, deeper networks showed a significant increase in explained variance which peaks at ∼200ms, but only within unmasked trials, not masked trials. In summary, we provided clear evidence that excitatory additive recurrent processing in ResNets captures some of the recurrent processing in humans.Significance statementThe challenge of modeling recurrent processes is not trivial and the operationalization of recurrent processing is highly contested. In this paper, we tested the ability of deep residual networks (ResNets) to explain recurrent processes in the human brain. Though ResNets are feedforward networks, they have been shown to equate operations in recurrent neural networks. In this study, we show that deeper networks explained more variance in brain activity than shallower networks. However, all networks still performed far from the noise ceiling. Thus, we conclude that recurrent processing in ResNets captures a form of recurrent processing in humans though other types of recurrent processing (inhibition, multiplicative) that are not present in current regular deep neural networks (alexnet, cornet, resnet) are necessary for building better visual models.

Aims Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro‐epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. Methods The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well‐characterised FCD 2 cohort. Results Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T‐lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. Conclusion We conclude that, next to mutation‐driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b., Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells in FCD 2b. We conclude that, next to mutation‐driven mTOR activation and seizure activity, balloon cells are crucial drivers of inflammation in FCD 2b.

Aims Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA‐34a (miR‐34a) among the most upregulated miRs in tubers. Here, we characterised miR‐34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR‐34a. Methods We analysed the expression of miR‐34a in resected cortical tubers (n = 37) compared with autopsy‐derived control tissue (n = 27). The effect of miR‐34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH‐SY5Y cells following miR‐34a transfection. Results The peak of miR‐34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR‐34a was also strongly expressed in foetal TSC cortex. Overexpression of miR‐34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR‐34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. Conclusions MicroRNA‐34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR‐34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC., MicroRNA‐34a (MiR‐34a) is overexpressed in cortical tubers from children with tuberous sclerosis complex with a peak during infancy. MiR‐34a negatively regulates constitutively activated mechanistic target of rapamycin complex 1 in human astrocytes. MiR‐34a overexpression impairs corticogenesis in the embryonic mouse brain, potentially through targeting genes involved in neuronal migration.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-021-02348-6.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Astrocytes are critical for healthy brain function. In Alzheimer’s disease, astrocytes become reactive, which affects their signaling properties. Here, we measured spontaneous calcium transients ex vivo in hippocampal astrocytes in brain slices containing the dentate gyrus of 6- (6M) and 9-month-old (9M) APPswe/PSEN1dE9 (APP/PS1) mice. We investigated the frequency and duration of calcium transients in relation to aging, amyloid-β (Aβ) pathology, and the proximity of the astrocyte to Aβ plaques. The 6M APP/PS1 astrocytes showed no change in spontaneous calcium-transient properties compared to wild-type (WT) astrocytes. 9M APP/PS1 astrocytes, however, showed more hyperactivity compared to WT, characterized by increased spontaneous calcium transients that were longer in duration. Our data also revealed an effect of aging, as 9M astrocytes overall showed an increase in calcium activity compared to 6M astrocytes. Subsequent calcium-wave analysis showed an increase in sequential calcium transients (i.e., calcium waves) in 9M astrocytes, suggesting increased network activity ex vivo. Further analysis using null models revealed that this network effect is caused by chance, due to the increased number of spontaneous transients. Our findings show that alterations in calcium signaling in individual hippocampal astrocytes of APP/PS1 mice are subject to both aging and Aβ pathology but these do not lead to a change in astrocyte network activity. These alterations in calcium dynamics of astrocytes may help to understand changes in neuronal physiology leading to cognitive decline and ultimately dementia.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of intra- and extracellular proteins. Several brain MMPs have been implicated in neurological disorders including epilepsy. We recently showed that the novel gelatinase inhibitor ACT-03 has disease-modifying effects in models of epilepsy. Here, we studied its effects on neuroinflammation and blood–brain barrier (BBB) integrity. Using the rapid kindling rat model of epilepsy, we examined whether ACT-03 affected astro- and microgliosis in the brain using immunohistochemistry. Cellular and molecular alterations were further studied in vitro using human fetal astrocyte and brain endothelial cell (hCMEC/D3) cultures, with a focus on neuroinflammatory markers as well as on barrier permeability using an endothelial and astrocyte co-culture model. We observed less astro- and microgliosis in the brains of kindled animals treated with ACT-03 compared to control vehicle-treated animals. In vitro, ACT-03 treatment attenuated stimulation-induced mRNA expression of several pro-inflammatory factors in human fetal astrocytes and brain endothelial cells, as well as a loss of barrier integrity in endothelial and astrocyte co-cultures. Since ACT-03 has disease-modifying effects in epilepsy models, possibly via limiting gliosis, inflammation, and barrier integrity loss, it is of interest to further evaluate its effects in a clinical trial.

Pattern separation is a hippocampal process in which highly similar stimuli are recognized as separate representations, and deficits could lead to memory impairments in neuropsychiatric disorders such as schizophrenia. The 5-HT1A receptor (5-HT1AR) is believed to be involved in these hippocampal pattern separation processes. However, in the dorsal raphe nucleus (DRN), the 5-HT1AR is expressed as a somatodendritic autoreceptor, negatively regulates serotonergic signaling, and could thereby counteract the effects of hippocampal postsynaptic 5-HT1A receptors. Therefore, this study aims to identify how pre- and post-synaptic 5-HT1AR activity affects pattern separation. Object pattern separation (OPS) performance was measured in male Wistar rats after both acute and chronic treatment (i.p.) with 5-HT1AR biased agonists F13714 (0.0025 mg/kg acutely, 0.02 mg/kg/day chronically) or NLX-101 (0.08 mg/kg acutely, 0.32 mg/kg/day chronically), which preferentially activate autoreceptors or postsynaptic receptors respectively, for 14 days. Body temperature - a functional correlate of hypothalamic 5-HT1AR stimulation - was measured daily. Additionally, 5-HT1AR density (DRN) and plasticity markers (hippocampus) were assessed. Acute treatment with F13714 impaired OPS performance, whereas chronic treatment normalized this, and a drop in body temperature was found from day 4 onwards. NLX-101 enhanced OPS performance acutely and chronically, and caused an acute drop in body temperature. Chronic NLX-101 treatment increased doublecortin positive neurons in the dorsal hippocampus, while chronic treatment with F13714 resulted in a downregulation of 5-HT1A autoreceptors, which likely reversed the acute impairment in OPS performance. Chronic treatment with NLX-101 appears to have therapeutic potential to improve brain plasticity and OPS performance.

Objective: Depressive disorders are common among about 50% of the patients with drug-resistant temporal lobe epilepsy (TLE). The underlying etiology remains elusive, but hypothalamus-pituitary-adrenal (HPA) axis activation due to changes in glucocorticoid receptor (GR) protein expression could play an important role. Therefore, we set out to investigate expression of the GR in the hippocampus, an important brain region for HPA axis feedback, of patients with drug-resistant TLE, with and without comorbid depression. Methods: GR expression was studied using immunohistochemistry on hippocampal sections from well-characterized TLE patients with depression (TLE + D, n = 14) and without depression (TLE − D, n = 12) who underwent surgery for drug-resistant epilepsy, as well as on hippocampal sections from autopsy control cases (n = 9). Video–electroencephalography (EEG), magnetic resonance imaging (MRI), and psychiatric and memory assessments were performed prior to surgery. Results: Abundant GR immunoreactivity was present in dentate gyrus granule cells and CA1 pyramidal cells of controls. In contrast, neuronal GR expression was lower in patients with TLE, particularly in the TLE + D group. Quantitative analysis showed a smaller GR+ area in TLE + D as compared to TLE − D patients and controls. Furthermore, the ratio between the number of GR+/NeuN+ cells was lower in patients with TLE + D as compared to TLE − D and correlated negatively with the depression severity based on psychiatric history. The expression of the GR was also lower in glial cells of TLE + D compared to TLE − D patients and correlated negatively to the severity of depression. Significance: Reduced hippocampal GR expression may be involved in the etiology of depression in patients with TLE and could constitute a biological marker of depression in these patients. Fil: D`alessio, Luciana. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Centro Universitario de Neurología "Dr. José María Ramos Mejía".; Argentina Fil: Mesarosova, Lucia. University of Amsterdam; Países Bajos Fil: Anink, Jasper J.. University of Amsterdam; Países Bajos Fil: Kochen, Sara Silvia. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Centro Universitario de Neurología "Dr. José María Ramos Mejía".; Argentina Fil: Solis, Patricia Cristina Lourdes. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Centro Universitario de Neurología "Dr. José María Ramos Mejía".; Argentina Fil: Oddo, Silvia Andrea. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Centro Universitario de Neurología "Dr. José María Ramos Mejía".; Argentina Fil: Konopka, Hector Felix. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Centro Universitario de Neurología "Dr. José María Ramos Mejía".; Argentina Fil: Iyer, Anand M.. University of Amsterdam; Países Bajos Fil: Mühlebner, Angelika. University of Amsterdam; Países Bajos Fil: Lucassen, Paul J.. University of Amsterdam; Países Bajos Fil: Aronica, Eleonora. University of Amsterdam; Países Bajos. Stichting Epilepsie Instellingen Nederland; Países Bajos Fil: van Vliet, Erwin A.. University of Amsterdam; Países Bajos

OBJECTIVE: Deep brain stimulation (DBS) is an increasingly applied treatment for various neuropsychiatric disorders including drug-resistant epilepsy, and it may be optimized by rationalizing the stimulation protocol based on increased knowledge of its mechanism of action. We evaluated the effects of minutes to hours of hippocampal DBS on hippocampal evoked potentials (EPs) and local field potentials (LFPs) in freely moving male rats to further investigate some of the previously proposed mechanisms of action.METHODS: Hippocampal high-frequency (130 Hz) DBS was administered for 0, 1, or 6 min every 10 min for 160 min. Stimulation parameter settings were similar to those that had previously been shown to reduce seizures in epileptic rats. EPs and LFPs were recorded in the stimulation-free intervals. We investigated both the immediate temporary effects of 1 or 6 min of DBS and the effects of 160 min of intermittent DBS. Input specificity was investigated by using two different stimulation electrodes.RESULTS: Relatively low DBS intensities corresponding to only 1.8% of the intensity evoking a maximum EP were required to prevent unintended seizure occurrence in healthy rats. Both 1 and 6 min of DBS caused input-specific short-lasting (P = .005). We observed longer-lasting, input-specific EP reductions during the 160 min intermittent DBS, with statistically significant reductions (3%-4%) of the fEPSP slope (P = .009-.018). The LFP spectrogram remained unaltered.SIGNIFICANCE: Deep brain stimulation induced both acute temporary effects compatible with axonal block and/or synaptic depression, and longer-lasting potentially cumulative EP reductions, suggesting the involvement of homeostatic plasticity or long-term depression. This dual time course may parallel the different temporal patterns of improvement observed in clinical trials. The longer-lasting reductions provide a potential neurophysiological basis for the use of intermittent DBS-as typically used in epilepsy patients-as an alternative to continuous DBS.

Epigenetics refers broadly to processes that influence medium to long-term gene expression by changing the readability and accessibility of the genetic code. The Neurobiology Commission of the International League Against Epilepsy (ILAE) recently convened a Task Force to explore and disseminate advances in epigenetics to better understand their role and intersection with genetics and the neurobiology of epilepsies and their co-morbidities, and to accelerate translation of these findings into the development of better therapies. Here, we provide a topic primer on epigenetics, explaining the key processes and findings to date in experimental and human epilepsy. We review the growing list of genes with epigenetic functions that have been linked with epilepsy in humans. We consider potential practical applications, including using epigenetic signals as biomarkers for tissue- and biofluid-based diagnostics and the prospects for developing epigenetic-based treatments for epilepsy. We include a glossary of terms, FAQs and other supports to facilitate a broad understanding of the topic for the non-expert. Last, we review the limitations, research gaps and the next challenges. In summary, epigenetic processes represent important mechanisms controlling the activity of genes, providing opportunities for insight into disease mechanisms, biomarkers and novel therapies for epilepsy.

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.

Tuberous sclerosis complex (TSC) is a congenital disorder characterized by cortical malformations and concomitant epilepsy caused by loss‐of‐function mutations in the mTOR suppressors TSC1 or TSC2. While the underlying molecular changes caused by mTOR activation in TSC have previously been investigated, the drivers of these transcriptional change have not been fully elucidated. A better understanding of the perturbed transcriptional regulation could lead to the identification of novel pathways for therapeutic intervention not only in TSC, but other genetic epilepsies in which mTOR activation plays a key role, such as focal cortical dysplasia 2b (FCD). Here, we analyzed RNA sequencing data from cortical tubers and a tsc2−/− zebrafish. We identified differential expression of the transcription factors (TFs) SPI1/PU.1, IRF8, GBX2, and IKZF1 of which SPI1/PU.1 and IRF8 targets were enriched among the differentially expressed genes. Furthermore, for SPI1/PU.1 these findings were conserved in TSC zebrafish model. Next, we confirmed overexpression of SPI1/PU.1 on the RNA and protein level in a separate cohort of surgically resected TSC tubers and FCD tissue, in fetal TSC tissue, and a Tsc1 GFAP−/− mouse model of TSC. Subsequently, we validated the expression of SPI1/PU.1 in dysmorphic cells with mTOR activation in TSC tubers. In fetal TSC, we detected SPI1/PU.1 expression prenatally and elevated RNA Spi1 expression in Tsc1 GFAP−/− mice before the development of seizures. Finally, in vitro, we identified that in astrocytes and neurons SPI1 transcription was driven by H2O2‐induced oxidative stress, independent of mTOR. We identified SPI1/PU.1 as a novel TF involved in the pro‐inflammatory gene expression of malformed cells in TSC and FCD 2b. This transcriptional program is activated in response to oxidative stress and already present prenatally. Importantly, SPI1/PU.1 protein appears to be strictly limited to malformed cells, as we did not find SPI1/PU.1 protein expression in mice nor in our in vitro models., Using RNA sequencing data from surgically resected tubers from tuberous sclerosis complex (TSC) patients and tissue from a tsc2−/− zebrafish knockout model we identified the transcription factor SPI1/PU.1 to be upregulated and its targets to be enriched among the differentially expressed genes. Furthermore, we validated these findings in a separate cohort of TSC as well as in the histopathologically similar focal cortical dysplasia (FCD) type 2b, in fetal TSC tissue and an experimental TSC model on the RNA and protein levels. We identified SPI1/PU.1 as a novel transcription factor involved in the pro‐inflammatory gene expression of malformed cells in TSC and FCD 2b and found in vitro evidence for its regulation by oxidative stress.

Contains fulltext : 239279.pdf (Publisher’s version ) (Open Access) The reproducibility crisis (or replication crisis) in biomedical research is a particularly existential and under-addressed issue in the field of behavioral neuroscience, where, in spite of efforts to standardize testing and assay protocols, several known and unknown sources of confounding environmental factors add to variance. Human interference is a major contributor to variability both within and across laboratories, as well as novelty-induced anxiety. Attempts to reduce human interference and to measure more "natural" behaviors in subjects has led to the development of automated home-cage monitoring systems. These systems enable prolonged and longitudinal recordings, and provide large continuous measures of spontaneous behavior that can be analyzed across multiple time scales. In this review, a diverse team of neuroscientists and product developers share their experiences using such an automated monitoring system that combines Noldus PhenoTyper® home-cages and the video-based tracking software, EthoVision® XT, to extract digital biomarkers of motor, emotional, social and cognitive behavior. After presenting our working definition of a "home-cage", we compare home-cage testing with more conventional out-of-cage tests (e.g., the open field) and outline the various advantages of the former, including opportunities for within-subject analyses and assessments of circadian and ultradian activity. Next, we address technical issues pertaining to the acquisition of behavioral data, such as the fine-tuning of the tracking software and the potential for integration with biotelemetry and optogenetics. Finally, we provide guidance on which behavioral measures to emphasize, how to filter, segment, and analyze behavior, and how to use analysis scripts. We summarize how the PhenoTyper has applications to study neuropharmacology as well as animal models of neurodegenerative and neuropsychiatric illness. Looking forward, we examine current challenges and the impact of new developments. Examples include the automated recognition of specific behaviors, unambiguous tracking of individuals in a social context, the development of more animal-centered measures of behavior and ways of dealing with large datasets. Together, we advocate that by embracing standardized home-cage monitoring platforms like the PhenoTyper, we are poised to directly assess issues pertaining to reproducibility, and more importantly, measure features of rodent behavior under more ethologically relevant scenarios. 26 p.

AIMS: Focal non-convulsive status epilepticus (FncSE) is a common emergency condition that may present as the first epileptic manifestation. In recent years, it has become increasingly clear that de novo FncSE should be promptly treated to improve post-status outcome. Whether seizure activity occurring during the course of the FncSE contributes to ensuing brain damage has not been demonstrated unequivocally and is here addressed.METHODS: We used continuous video-EEG monitoring to characterise an acute experimental FncSE model induced by unilateral intrahippocampal injection of kainic acid (KA) in guinea pigs. Immunohistochemistry and mRNA expression analysis were utilised to detect and quantify brain injury, 3-days and 1-month after FncSE.RESULTS: Seizure activity occurring during the course of FncSE involved both hippocampi equally. Neuronal loss, blood-brain barrier permeability changes, gliosis and up-regulation of inflammation, activity-induced and astrocyte-specific genes were observed in the KA-injected hippocampus. Diazepam treatment reduced FncSE duration and KA-induced neuropathological damage. In the contralateral hippocampus, transient and possibly reversible gliosis with increase of aquaporin-4 and Kir4.1 genes were observed 3 days post-KA. No tissue injury and gene expression changes were found 1-month after FncSE.CONCLUSIONS: In our model, focal seizures occurring during FncSE worsen ipsilateral KA-induced tissue damage. FncSE only transiently activated glia in regions remote from KA-injection, suggesting that seizure activity during FncSE without local pathogenic co-factors does not promote long-lasting detrimental changes in the brain. These findings demonstrate that in our experimental model, brain damage remains circumscribed to the area where the primary cause (KA) of the FncSE acts. Our study emphasises the need to use antiepileptic drugs to contain local damage induced by focal seizures that occur during FncSE.

Background: Glycogen synthase kinase-3β (GSK3β) is a key regulator of cellular homeostasis. In neurons, GSK3β contributes to the control of neuronal transmission and plasticity, but its role in epilepsy remains to be defined. Methods: Biochemical and electrophysiological methods were used to assess the role of GSK3β in regulating neuronal transmission and epileptogenesis. GSK3β activity was increased genetically in GSK3β[S9A] mice. Its effects on neuronal transmission and epileptogenesis induced by kainic acid were assessed by field potential recordings in mice brain slices and video electroencephalography in vivo. The ion channel expression was measured in brain samples from mice and followed by analysis in samples from patients with temporal lobe epilepsy or focal cortical dysplasia in correlation to GSK3β phosphorylation. Findings: Higher GSK3β activity decreased the susceptibility to kainic acid-induced epileptiform discharges and the progression of epileptogenesis. At the biochemical level, higher GSK3β activity increased the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 4 and decreased phosphorylation of the glutamate α±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1. The phosphorylation of GluA1 at Serine 831 and Serine 845 was lower in GSK3β[S9A] mice than in wildtype controls, under basal conditions and in the epileptic mouse brain, respectively. Moreover, we found a significant correlation between higher inhibitory GSK3β phosphorylation at Serine 9 and higher activating GluA1 phosphorylation at Serine 845 in brain samples from epileptic patients. Interpretation: Our data imply GSK3β activity in the protection of neuronal networks from hyper-activation and indicate that the anti-epileptogenic function of GSK3I² involves decreasing the synaptic AMPA receptors pool. Funding: Research was supported by the Polish National Science Centre (no. 2011/01/N/NZ3/05409 to MU; no. 2017/26/D/NZ4/00159 to PK-G) and FP7 European Union grants (no. 223276, “NeuroGSK3” to FvL and JJ; no. 229676, “HealthProt” to JJ; no. 602102, “Epitarget” to EV and EA.). This study was also co-financed by European Union funds from the European Social Fund (MU). The work of MU was partially financed by IUVENTUS IP2012 037872. The work of MU, KKotulska, SJ, EA and JJ was partly supported by 7FP grant “EPISTOP” (grant agreement no. 602391), and the Polish Ministerial funds for science (years 2014-2019) for the implementation of international co-financed projects. The work of AG was financed by the Polish National Science Centre (grant no. 2011/01/B/NZ3/05397). KKotulska, SJ and JJ were partly financed by the Polish National Center for Research and Development (grant EPIMARKER, no. STRATEGMED3/306306/4/2017). MU was a recipient of a L’OrealUNESCO for Women and Science Fellowship in Poland and EMBO Short Term Fellowship. JJ and MU were also recipients of a Foundation for Polish Science "Mistrz" Professorial Subsidy and Fellowship, respectively. Declaration of Interest: SJ received Advisory Board honoraria from Novartis Oncology. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical Approval: The study was approved by the local Ethics Board in Warsaw (approval no. 223/KBE/2015).

ObjectiveTo identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to control patients with epilepsy.MethodsFor proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected postmortem brain tissue of 12 patients with SUDEP and 14 with non-SUDEP epilepsy. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from patients with mesial temporal lobe epilepsy: 6 low-risk and 8 high-risk SUDEP as determined by a short (ResultsIn autopsy hippocampus and cortex, we observed no proteomic differences between patients with SUDEP and those with non-SUDEP epilepsy, contrasting with our previously reported robust differences between epilepsy and controls without epilepsy. Transcriptomics in hippocampus and cortex from patients with surgical epilepsy segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 long noncoding RNAs, 3 pending) in hippocampus.ConclusionThe SUDEP proteome and high-risk SUDEP transcriptome were similar to those in other patients with epilepsy in hippocampus and cortex, consistent with diverse epilepsy syndromes and comorbid conditions associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions, may identify molecular mechanisms of SUDEP.

Matrix metalloproteinases (MMPs) are synthesized by neurons and glia and released into the extracellular space, where they act as modulators of neuroplasticity and neuroinflammatory agents. Development of epilepsy (epileptogenesis) is associated with increased expression of MMPs, and therefore, they may represent potential therapeutic drug targets. Using quantitative PCR (qPCR) and immunohistochemistry, we studied the expression of MMPs and their endogenous inhibitors tissue inhibitors of metalloproteinases (TIMPs) in patients with status epilepticus (SE) or temporal lobe epilepsy (TLE) and in a rat TLE model. Furthermore, we tested the MMP2/9 inhibitor IPR-179 in the rapid-kindling rat model and in the intrahippocampal kainic acid mouse model. In both human and experimental epilepsy, MMP and TIMP expression were persistently dysregulated in the hippocampus compared with in controls. IPR-179 treatment reduced seizure severity in the rapid-kindling model and reduced the number of spontaneous seizures in the kainic acid model (during and up to 7 weeks after delivery) without side effects while improving cognitive behavior. Moreover, our data suggest that IPR-179 prevented an MMP2/9-dependent switch-off normally restraining network excitability during the activity period. Since increased MMP expression is a prominent hallmark of the human epileptogenic brain and the MMP inhibitor IPR-179 exhibits antiseizure and antiepileptogenic effects in rodent epilepsy models and attenuates seizure-induced cognitive decline, it deserves further investigation in clinical trials.

The blood-brain barrier (BBB) is important to maintain brain homeostasis, which is crucial for the functionality of neuronal networks. BBB dysfunction is observed in several neurological diseases, including epilepsy. Prolonged BBB dysfunction and subsequent perivascular inflammation as well as alterations in the extracellular matrix (ECM) play an important role in epileptogenesis. In this chapter we aim to give an overview of the processes involved in BBB dysfunction and epileptogenesis and provide evidence from animal as well as from human studies. We will focus on modulators of BBB function including specific cell types, inflammatory mediators as well as ECM molecules. Furthermore, we will discuss new therapeutic targets and the results of intervention studies that are important for the development of novel therapeutic approaches in epilepsy.

There is growing evidence that mechanical factors affect brain functioning. However, brain components responsible for regulating the physiological mechanical environment are not completely understood. To determine the relationship between structure and stiffness of brain tissue, we performed high-resolution viscoelastic mapping by dynamic indentation of the hippocampus and the cerebellum of juvenile mice brains, and quantified relative area covered by neurons (NeuN-staining), axons (neurofilament NN18-staining), astrocytes (GFAP-staining), myelin (MBP-staining) and nuclei (Hoechst-staining) of juvenile and adult mouse brain slices. Results show that brain subregions have distinct viscoelastic parameters. In gray matter (GM) regions, the storage modulus correlates negatively with the relative area of nuclei and neurons, and positively with astrocytes. The storage modulus also correlates negatively with the relative area of myelin and axons (high cell density regions are excluded). Furthermore, adult brain regions are ∼ 20%-150% stiffer than the comparable juvenile regions which coincide with increase in astrocyte GFAP-staining. Several linear regression models are examined to predict the mechanical properties of the brain tissue based on (immuno)histochemical stainings.

Tuberous sclerosis complex (TSC), a rare genetic disorder caused by a mutation in the TSC1 or TSC2 gene, is characterized by the growth of hamartomas in several organs. This includes the growth of low-grade brain tumors, known as subependymal giant cell astrocytomas (SEGA). Previous studies have shown differential expression of genes related to the extracellular matrix in SEGA. Matrix metalloproteinases (MMPs), and their tissue inhibitors (TIMPs) are responsible for remodeling the extracellular matrix and are associated with tumorigenesis. This study aimed to investigate the MMP/TIMP proteolytic system in SEGA and the regulation of MMPs by microRNAs, which are important post-transcriptional regulators of gene expression. We investigated the expression of MMPs and TIMPs using previously produced RNA-Sequencing data, real-time quantitative PCR and immunohistochemistry in TSC-SEGA samples and controls. We found altered expression of several MMPs and TIMPs in SEGA compared to controls. We identified the lowly expressed miR-320d in SEGA as a potential regulator of MMPs, which can decrease MMP2 expression in human fetal astrocyte cultures. This study provides evidence of a dysregulated MMP/TIMP proteolytic system in SEGA of which MMP2 could be rescued by microRNA-320d. Therefore, further elucidating microRNA-mediated MMP regulation may provide insights into SEGA pathogenesis and identify novel therapeutic targets., peer-reviewed

Traumatic brain injury (TBI) dysregulates microRNAs, which are the master regulators of gene expression. Here we investigated the changes in a brain-enriched miR-124-3p, which is known to associate with major post-injury pathologies, such as neuroinflammation. RT-qPCR of the rat tissue sampled at 7 d and 3 months in the perilesional cortex adjacent to the necrotic lesion core (aPeCx) revealed downregulation of miR-124-3p at 7 d (fold-change (FC) 0.13, p < 0.05 compared with control) and 3 months (FC 0.40, p < 0.05) post-TBI. In situ hybridization confirmed the downregulation of miR-124-3p at 7 d and 3 months post-TBI in the aPeCx (both p < 0.01). RT-qPCR confirmed the upregulation of the miR-124-3p target Stat3 in the aPeCx at 7 d post-TBI (7-fold, p < 0.05). mRNA-Seq revealed 312 downregulated and 311 upregulated miR-124 targets (p < 0.05). To investigate whether experimental findings translated to humans, we performed in situ hybridization of miR-124-3p in temporal lobe autopsy samples of TBI patients. Our data revealed downregulation of miR-124-3p in individual neurons of cortical layer III. These findings indicate a persistent downregulation of miR-124-3p in the perilesional cortex that might contribute to post-injury neurodegeneration and inflammation.

Neurons in many brain regions exhibit spontaneous, intrinsic rhythmic firing activity. This rhythmic firing activity may determine the way in which these neurons respond to extrinsic synaptic inputs. We hypothesized that neurons should be most responsive to inputs at the frequency of the intrinsic oscillation frequency. We addressed this question in the ventral tegmental area (VTA), a dopaminergic nucleus in the midbrain. VTA neurons have a unique propensity to exhibit spontaneous intrinsic rhythmic activity in the 1-5 Hz frequency range, which persists in the in-vitro brain slice, and form a network of weakly coupled oscillators. Here, we combine in-vitro simultaneous recording of action potentials from a 60 channel multi-electrode-array with cell-type-specific optogenetic stimulation of the VTA dopamine neurons. We investigated how VTA neurons respond to wide-band stochastic (Poisson input) as well as regular laser pulses. Strong synchrony was induced between the laser input and the spike timing of the neurons, both for regular pulse trains and Poisson pulse trains. For rhythmically pulsed input, the neurons demonstrated resonant behavior with the strongest phase locking at their intrinsic oscillation frequency, but also at half and double the intrinsic oscillation frequency. Stochastic Poisson pulse stimulation provided a more effective stimulation of the entire population, yet we observed resonance at lower frequencies (approximately half the oscillation frequency) than the neurons' intrinsic oscillation frequency. The non-linear filter characteristics of dopamine neurons could allow the VTA to predict precisely timed future rewards based on past sensory inputs, a crucial component of reward prediction error signaling. In addition, these filter characteristics could contribute to a pacemaker role for the VTA in synchronizing activity with other regions like the prefrontal cortex and the hippocampus.

The adult human brain contains over 100 billion neurons, and each neuron is interconnected with other neurons by thousands of contact points called synapses. The strength of these synapses can change, and it is this phenomenon, called synaptic plasticity, that underlies the capacity of experiences to remodel neural circuitry, enabling adaptive behavior, learning and memory. However, for many cases in which synaptic plasticity occurs, the underlying cellular mechanism is still unclear and new tools to study the synaptic changes underlying synaptic plasticity are still required. In this thesis, I validated the variance-to-mean ratio (VMR) as a tool to study the mechanisms underlying synaptic plasticity. The VMR is a statistical tool based on the quantal model of synaptic transmission, and can be used in combination with the conventional quantal measure of the inverse square of the coefficient of variation (1/CV2). I found that calculating both 1/CV2 and VMR values of evoked synaptic currents before and after an alteration in synaptic strength allows for a fast and reliable prediction of the source of synaptic plasticity. I then used this variance analysis in combination with other experimental tools to investigate the organization of AMPA-receptor and NMDA-receptor subtypes at synapses onto hippocampal CA1 pyramidal neurons that receive Schaffer collateral input from CA3 neurons, and to investigate multiple types of synaptic plasticity, such as amyloid-β-induced synaptic depression, a phenomenon that is thought to be crucially involved in Alzheimer’s disease, and β-adrenergic activation-induced synaptic potentiation.

BackgroundThe strength of synaptic transmission onto a neuron depends on the number of functional vesicle release sites (N), the probability of vesicle release (Pr), and the quantal size (Q). Statistical tools based on the quantal model of synaptic transmission can be used to acquire information on which of these parameters is the source of plasticity. However, quantal analysis depends on assumptions that may not be met at central synapses.New methodWe examined the merit of quantal analysis to extract the mechanisms underlying synaptic plasticity by applying binomial statistics on the variance in amplitude of postsynaptic currents evoked at Schaffer collateral-CA1 (Sc-CA1) synapses in mouse hippocampal slices. We extend this analysis by combining the conventional inverse square of the coefficient of variation (1/CV2) with the variance-to-mean ratio (VMR).ResultsThis method can be used to assess the relative, but not absolute, contribution of N, Pr and Q to synaptic plasticity. The changes in 1/CV2 and VMR values correctly reflect experimental modifications of N, Pr and Q at Sc-CA1 synapses.Comparison with existing methodsWhile the 1/CV2 depends on N and Pr, but is independent of Q, the VMR is dependent on Pr and Q, but not on N. Combining both allows for a rapid assessment of the mechanism underlying synaptic plasticity without the need for additional electrophysiological experiments.ConclusionCombining the 1/CV2 with the VMR allows for a reliable prediction of the relative contribution of changes in N, Pr and Q to synaptic plasticity.

In this thesis, we investigated the impact of Alzheimer’s disease on neuronal function and glial cells. Hereto we used various models, i.e. the APPswePS1dE9 AD mouse model, human post-mortem brain material, and a microglia-like cell model. In a review we focused on the role of reactive astrocytes in the AD mouse model, indicating that reactive astrocytes can be a promising treatment target in AD. Using the same mouse model, we investigated changes in neuronal function at various ages. We concluded that aging has a more prominent effect on the measurements than the increased presence of amyloid. In a first experiment, to investigate the potential role of microglia in AD, we used a microglia-like model to examine the effect of Aβ1-42 oligomers on transcriptomic changes. Interestingly, we observed a distinct upregulation of metallothionein subtypes in the Aβ1-42 oligomer stimulated cells. The upregulation of metallothioneins could have a neuroprotective function against the oxidative stress and neuroinflammation involved in AD, indicating that the acute activation by Aβ1-42 oligomers could induce a protective response. We characterized the Aβ plaque burden and morphology in cortical brain slices of AD patients and non-demented control with amyloid plaques. Even though the last group has overall less Aβ plaques, relatively more dense-core plaques were observed. We found no differences in the total number of microglia or morphology between the groups. Further characterization of plaque-associated microglia suggested a reduced phagocytic activity by microglia surrounding dense-core plaques in NDC+-cases. No difference in synaptic density between AD- and NDC+-cases near dense-core or diffuse plaques was detected.

Objective: Dravet syndrome is a rare neurodevelopmental disease, characterized by general cognitive impairment and severe refractory seizures. The majority of patients carry the gene mutation SCN1A, leading to a defective sodium channel that contributes to pathogenic brain excitability. A γ‐aminobutyric acid (GABAergic) impairment, as in other neurodevelopmental diseases, has been proposed as an additional mechanism, suggesting that seizures could be alleviated by GABAergic therapies. However, up to now the physiological mechanisms underlying the GABAergic dysfunction in Dravet syndrome are still unknown due to the scarce availability of this brain tissue. Here we studied, for the first time, human GABAA‐evoked currents using cortical brain tissue from Dravet syndrome patients.Methods: We transplanted in Xenopus oocytes cell membranes obtained from brain tissues of autopsies of Dravet syndrome patients, tuberous sclerosis complex patients as a pathological comparison, and age‐matched controls. Additionally, experiments were performed on oocytes expressing human α1β2γ2 and α1β2 GABAA receptors. GABAA currents were recorded using the two‐microelectrodes voltage‐clamp technique. Quantitative real‐time polymerase chain reaction, immunohistochemistry, and double‐labeling techniques were carried out on the same tissue samples. Results: We found (1) a decrease in GABA sensitivity in Dravet syndrome compared to controls, which was related to an increase in α4‐ relative to α1‐containing GABAA receptors; (2) a shift of the GABA reversal potential toward more depolarizing values in Dravet syndrome, and a parallel increase of the chloride transporters NKCC1/KCC2 expression ratio; (3) an increase of GABAA currents induced by low doses of cannabidiol both in Dravet syndrome and tuberous sclerosis complex comparable to that induced by a classical benzodiazepine, flunitrazepam, that still persists in γ‐less GABAA receptors.Significance: Our study indicates that a dysfunction of the GABAergic system, considered as a feature of brain immaturity, together with defective sodium channels, can contribute to a general reduction of inhibitory efficacy in Dravet brain, suggesting that GABAA receptors could be a target for new therapies.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force created the TASK3 working groups to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve standardization of experimental designs. This article concerns the parameters that can be measured to assess the physiologic condition of the animals that are used to study rodent models of epilepsy. Here we discuss CDEs for physiologic parameters measured in adult rats and mice such as general health status, temperature, cardiac and respiratory function, and blood constituents. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript we discuss the monitoring of different aspects of physiology of the animals. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of biomarkers and new treatments for epilepsy.

OBJECTIVE: Because brain inflammation may contribute to the pathophysiology of temporal lobe epilepsy (TLE), we investigated the expression of various inflammatory markers of the innate and adaptive immune system in the epileptogenic human and rat hippocampus in relation to seizure activity and blood-brain barrier (BBB) dysfunction.METHODS: Immunohistochemistry was performed using various immune cell markers (for microglia, monocytes, macrophages, T lymphocytes, and dendritic cells) on hippocampal sections of drug-resistant TLE patients and patients who died after status epilepticus. The expression of these markers was also studied in the electrical post-status epilepticus rat model for TLE, during the acute, latent, and chronic epileptic phase. BBB dysfunction was assessed using albumin immunohistochemistry and the BBB tracer fluorescein.RESULTS: Monocyte infiltration, microglia, and perivascular macrophage activation were persistently increased in both epileptogenic human and rat hippocampus, whereas T lymphocytes and dendritic cells were not or were scarcely detected. In addition to this, increased expression of C-C motif ligand 2 (CCL2) and osteopontin was observed. In humans, the expression of CD68 and CCL2 was related to the duration of epilepsy and type of pathology. In rats, the expression of CD68, CCL2, and the perivascular macrophage marker CD163 was related to the duration of the initial insult and to the number of spontaneous seizures. Interestingly, the number of CD163-positive perivascular macrophages was also positively correlated to BBB dysfunction in chronic epileptic rats.SIGNIFICANCE: These data suggest a proepileptogenic role for monocytes/macrophages and other cells of the innate immune response, possibly via increased BBB leakage, and indicate that T cells and dendritic cells, which are closely associated with the adaptive immune response, are only sparsely infiltrated during epileptogenesis in the electrical post-status epilepticus rat model. Future studies should reveal the relative importance of these immune cells and whether specific manipulation can modify or prevent epileptogenesis.

People that committed suicide were reported to have enhanced levels of gene transcripts for synaptic proteins in their prefrontal cortex (PFC). Given the close association of suicide with major depressive disorder (MDD), we here assessed whether these changes are related to suicide or rather to depression per se. We used quantitative PCR to determine mRNA levels of 32 genes encoding for proteins directly involved in glutamatergic or GABAergic synaptic transmission in postmortem samples of the anterior cingulate cortex (ACC) and the dorsolateral PFC (DLPFC). Seventy-two brain samples from 3 groups of subjects were derived from the Stanley Medical Research Institute (SMRI): i) patients with MDD who committed suicide (MDD-S), ii) MDD patients who died of non-suicidal causes (MDD-NS) and iii) age-matched, non-psychiatric control subjects. In the ACC, a significantly enhanced expression of genes related to glutamatergic or GABAergic synaptic transmission was found only in MDD-S patients, whereas in MDD-NS patients, decreased levels for these transcripts were found. Moreover, in the DLPFC, expression of these genes was decreased in MDD-S, relative to MDD-NS patients, whereas both groups showed increased expression compared to control subjects. In conclusion, our findings indicate that MDD is associated with increases in GABA and glutamate related genes in the DLPFC (irrespective of suicide), while in the ACC, the increase in GABA and glutamate related genes may relate to suicide, rather than to MDD per se.

Astrocytes are important mediators of inflammatory processes in the brain and seem to play an important role in several neurological disorders, including epilepsy. Recent studies show that astrocytes produce several microRNAs, which may function as crucial regulators of inflammatory pathways and could be used as therapeutic target. We aim to study which miRNAs are produced by astrocytes during IL-1β mediated inflammatory conditions in vitro, as well as their functional role and to validate these findings in human epileptogenic brain tissue. Sequencing was used to assess miRNA and mRNA expression in IL-1β-stimulated human fetal astrocyte cultures. miRNAs were overexpressed in cell cultures using miRNA mimics. Expression of miRNAs in resected brain tissue from patients with tuberous sclerosis complex or temporal lobe epilepsy with hippocampal sclerosis was examined using in situ hybridization. Two differentially expressed miRNAs were found: miR146a and miR147b, which were associated with increased expression of genes related to the immune/inflammatory response. As previously reported for miR146a, overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 and COX-2 after IL-1β stimulation in both astrocyte and tuberous sclerosis complex cell cultures. miR146a and miR147b overexpression decreased proliferation of astrocytes and promoted neuronal differentiation of human neural stem cells. Similarly to previous evidence for miR146a, miR147b was increased expressed in astrocytes in epileptogenic brain. Due to their anti-inflammatory effects, ability to restore aberrant astrocytic proliferation and promote neuronal differentiation, miR146a and miR147b deserve further investigation as potential therapeutic targets in neurological disorders associated with inflammation, such as epilepsy.

The perirhinal (PER) and lateral entorhinal (LEC) cortex form an anatomical link between the neocortex and the hippocampus. However, neocortical activity is transmitted through the PER and LEC to the hippocampus with a low probability, suggesting the involvement of the inhibitory network. This study explored the role of interneuron mediated inhibition, activated by electrical stimulation in the agranular insular cortex (AiP), in the deep layers of the PER and LEC. Activated synaptic input by AiP stimulation rarely evoked action potentials in the PER-LEC deep layer excitatory principal neurons, most probably because the evoked synaptic response consisted of a small excitatory and large inhibitory conductance. Furthermore, parvalbumin positive (PV) interneurons - a subset of interneurons projecting onto the axo-somatic region of principal neurons - received synaptic input earlier than principal neurons, suggesting recruitment of feedforward inhibition. This synaptic input in PV interneurons evoked varying trains of action potentials, explaining the fast rising, long lasting synaptic inhibition received by deep layer principal neurons. Altogether, the excitatory input from the AiP onto deep layer principal neurons is overruled by strong feedforward inhibition. PV interneurons, with their fast, extensive stimulus-evoked firing, are able to deliver this fast evoked inhibition in principal neurons. This indicates an essential role for PV interneurons in the gating mechanism of the PER-LEC network.

Article, Lack of translation of data obtained in preclinical trials to clinic has kindled researchers to develop new methodologies to increase the power and reproducibility of preclinical studies. One approach relates to harmonization of data collection and analysis, and has been used for a long time in clinical studies testing anti-seizure drugs. EPITARGET is a European Union FP7-funded research consortium composed of 18 partners from 9 countries. Its main research objective is to identify biomarkers and develop treatments for epileptogenesis. As the first step of harmonization of procedures between laboratories, EPITARGET established working groups for designing project-tailored common data elements (CDEs) and case report forms (CRFs) to be used in data collection and analysis. Eight major modules of CRFs were developed, presenting >1000 data points for each animal. EPITARGET presents the first single-project effort for harmonization of preclinical data collection and analysis in epilepsy research. EPITARGET is also anticipating the future challenges and requirements in a larger-scale preclinical harmonization of epilepsy studies, including training, data management expertise, cost, location, data safety and continuity of data repositories during and after funding period, and incentives motivating for the use of CDEs., final draft, http://purl.org/eprint/status/PeerReviewed

Understanding the relation between (sensory) stimuli and the activity of neurons (i.e., "the neural code") lies at heart of understanding the computational properties of the brain. However, quantifying the information between a stimulus and a spike train has proven to be challenging. We propose a new (in vitro) method to measure how much information a single neuron transfers from the input it receives to its output spike train. The input is generated by an artificial neural network that responds to a randomly appearing and disappearing "sensory stimulus": the hidden state. The sum of this network activity is injected as current input into the neuron under investigation. The mutual information between the hidden state on the one hand and spike trains of the artificial network or the recorded spike train on the other hand can easily be estimated due to the binary shape of the hidden state. The characteristics of the input current, such as the time constant as a result of the (dis)appearance rate of the hidden state or the amplitude of the input current (the firing frequency of the neurons in the artificial network), can independently be varied. As an example, we apply this method to pyramidal neurons in the CA1 of mouse hippocampi and compare the recorded spike trains to the optimal response of the "Bayesian neuron" (BN). We conclude that like in the BN, information transfer in hippocampal pyramidal cells is non-linear and amplifying: the information loss between the artificial input and the output spike train is high if the input to the neuron (the firing of the artificial network) is not very informative about the hidden state. If the input to the neuron does contain a lot of information about the hidden state, the information loss is low. Moreover, neurons increase their firing rates in case the (dis)appearance rate is high, so that the (relative) amount of transferred information stays constant.

AIMS: Oxidative stress is evident in resected epileptogenic brain tissue of patients with developmental brain malformations related to mammalian target of rapamycin activation: tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCD IIb). Whether chronic activation of anti-oxidant pathways is beneficial or contributes to pathology is not clear.METHODS: We investigated oxidative stress markers, including haem oxygenase 1, ferritin and the inflammation associated microRNA-155 in surgically resected epileptogenic brain tissue of TSC (n = 10) and FCD IIb (n = 8) patients and in a TSC model (Tsc1GFAP-/- mice) using immunohistochemistry, in situ hybridization, real-time quantitative PCR and immunoblotting. Using human foetal astrocytes we performed an in vitro characterization of the anti-oxidant response to acute and chronic oxidative stress and evaluated overexpression of the disease-relevant pro-inflammatory microRNA-155.RESULTS: Resected TSC or FCD IIb tissue displayed higher expression of oxidative stress markers and microRNA-155. Tsc1GFAP-/- mice expressed more microRNA-155 and haem oxygenase 1 in the brain compared to wild-type, preceding the typical development of spontaneous seizures in these animals. In vitro, chronic microRNA-155 overexpression induced haem oxygenase 1, iron regulatory elements and increased susceptibility to oxidative stress. Overexpression of iron regulatory genes was also detected in patients with TSC, FCD IIb and Tsc1GFAP-/- mice.CONCLUSION: Our results demonstrate that early and sustained activation of anti-oxidant signalling and dysregulation of iron metabolism are a pathological hallmark of FCD IIb and TSC. Our findings suggest novel therapeutic strategies aimed at controlling the pathological link between both processes.

Micro Electrode Arrays were used to simultaneously record spontaneous extracellular action potentials from 10 to 30 dopamine neurons in acute brain slices from the lateral Ventral Tegmental Area (VTA) of the rat. The spike train of an individual neuron was used to characterize the firing pattern: firing rate, firing irregularity and oscillation frequency. Functional connectivity between a pair of neurons was quantified by the Paired Phase Consistency (PPC), taking the oscillation frequency as reference. Under baseline conditions the PPC was significantly different from zero and 42 of the 386 pairs of VTA neurons showed significant coupling. Fifty percent of the recorded dopamine neurons were part of the coupled VTA network. Raising extracellular potassium from 3.5 to 5 mM increased the mean firing rate of the dopamine neurons by 45%. The same increase could be induced by bath application of 300 μm glutamate. High potassium reduced the PPC, but it did not change during the glutamate application. Our findings imply that manipulating excitability has distinct and specific consequences for functional connectivity in the VTA network that cannot be directly predicted from the changes in neuronal firing rates. Functional connectivity reflects the spatial organization and synchronization of the VTA output and thus represents a unique element of the message that is sent to the mesolimbic projection area. It adds a dimension to pharmacological manipulation of the VTA micro circuit that might help to understand the pharmacological (side) effects of e.g., anti-psychotic drugs.

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H2O2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100 µM, but not 50 µM, of H2O2 increased expression of TLR4, IL-1β and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100 µM H2O2 leading to upregulation of TLR4 signaling and IL-1β. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once.

OBJECTIVEInhibition of the mammalian target of rapamycin (mTOR) pathway could be antiepileptogenic in temporal lobe epilepsy (TLE), possibly via anti-inflammatory actions. We studied effects of the mTOR inhibitor rapamycin and the anti-inflammatory compound curcumin-also reported to inhibit the mTOR pathway-on epileptogenesis and inflammation in an in vitro organotypic hippocampal-entorhinal cortex slice culture model.METHODS Brain slices containing hippocampus and entorhinal cortex were obtained from 6-day-old rat pups and maintained in culture for up to 3 weeks. Rapamycin or curcumin was added to the culture medium from day 2 in vitro onward. Electrophysiological recordings revealed epileptiformlike activity that developed over 3 weeks.RESULTSIn week 3, spontaneous seizurelike events (SLEs) could be detected using whole cell recordings from CA1 principal neurons. The percentage of recorded CA1 neurons displaying SLEs was lower in curcumin-treated slice cultures compared to vehicle-treated slices (25.8% vs 72.5%), whereas rapamycin did not reduce SLE occurrence significantly (52%). Western blot for phosphorylated-S6 (pS6) and phosphorylated S6K confirmed that rapamycin inhibited the mTOR pathway, whereas curcumin only lowered pS6 expression at one phosphorylation site. Real-time quantitative polymerase chain reaction results indicated a trend toward lower expression of inflammatory markers IL-1β and IL-6 and transforming growth factor β after 3 weeks of treatment with rapamycin and curcumin compared to vehicle.SIGNIFICANCEOur results show that curcumin suppresses SLEs in the combined hippocampal-entorhinal cortex slice culture model and suggest that its antiepileptogenic effects should be further investigated in experimental models of TLE.

AIM Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain.METHODS We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures.RESULTSWe demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1β-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures.CONCLUSIONSThis study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

Virtually all brain functions, including the formation of new memories, require communication between braincells via their specialized contacts called ‘synapses’. Synapses use various AMPA-receptors to facilitate the communication between braincells, such as GluA1 or GluA3 AMPA-receptors. Impaired synapse function can lead to cognitive decline like in Alzheimer’s disease, where the accumulation of amyoid beta (aβ) damages synapses. This thesis is dedicated to the question of how aβ accumulation affects synapses and how this can be prevented. Chapter 1 : Demonstrates that GluA3 deficiency effectively protects against the negative effects of aβ on synapses, memory and life expectancy of Alzheimer mouse models. Chapter 2 : Synaptic GluA3 AMPA-receptors exist in active and in-activated states. Activation of GluA3 AMPA-receptors regulates synaptic strength, constituting synaptic plasticity. GluA3 AMPA-receptors can be activated by a noradrenaline induced rise in cAMP. Chapter 3 : Preventing the aβ induced removal of synaptic GluA3 AMPA-receptors, protects synapses against the effects of aβ. Chapter 4 : Sindbis-virus induced protein expression is a valid tool to study the physiology of living neurons (type of brain-cell). The research described in thesis reveals a biochemical process which, when targeted by therapies, can help protect against cognitive symptoms in Alzheimer disease. One such therapy could involve reducing synaptic GluA3 with frequent behavioural and cognitive exercises.

The research described in this dissertation offers insight in how transmission of neuronal activity is regulated in the perirhinal-entorhinal cortex, a brain area involved in memory processing. The perirhinal-entorhinal cortex is anatomically separated into an input and output network via the superficial and deep layers, respectively. This separation promotes that cortical activity is transmitted through the PER-LEC superficial layers towards the hippocampus for further processing. Via the activation of deep layer inhibitory interneurons, the output pathway of the hippocampus is blocked when activity is transmitted towards the hippocampus. It is known that emotion can modulate memory storage and retrieval, which increases the chances for survival. The amygdala, a structure involved in emotion, can modulate the transmission of activity through the perirhinal-entorhinal cortical network and herewith regulate memory formation. Although it has long been thought that synaptic input from the LA and cortex mainly interacted on the excitatory synaptic responses, we showed in this dissertation that modulation of inhibition probably plays a crucial role in the regulation of transmission by the amygdala. Co-activation of the amygdala induced prompter action potential firing of the inhibitory neurons, which probably ensures synchronization of firing of principal neurons. This synchronization caused by emotional modulation is suggested to ensure efficient and time-delimited information transmission to the projection areas.

Epileptogenesis is the collective of processes that underlie the transition of a healthy neural network into a network that can generate epileptic seizures. In Temporal Lobe Epilepsy (TLE), epileptogenesis is often associated with cell loss, inflammation, oxidative stress, and axonal sprouting in the hippocampus. Also, hyper-activation of the mammalian target of rapamycin (mTOR) pathway is thought to be involved in epileptogenesis. Interfering with mTOR activation or epileptogenic processes such as inflammation could reduce or prevent epileptogenesis, and thus lead to the development of anti-epileptogenic treatments for TLE. This thesis investigates epileptogenesis and the role of the mTOR pathway in TLE, and the anti-epileptogenic potential of two compounds: rapamycin and curcumin, using several experimental models for TLE in vivo and in vitro. Rapamycin, the main inhibitor of the mTOR pathway, was found to suppress epilepsy in vivo, but it did not completely reverse or prevent epileptogenesis. Also, rapamycin treatment was associated with side effects on growth. As an alternative mTOR inhibitor, curcumin, the anti-oxidant and anti-inflammatory component of turmeric, was investigated. Whereas effects of rapamycin were strongly related to mTOR inhibition, experiments in vitro demonstrated that curcumin rather targeted inflammatory pathways. Curcumin reduced epileptogenesis in vitro, but its low bioavailability likely limited its efficacy in vivo. Although this suggests that the road towards a safe and effective anti-epileptogenic treatment is long, this thesis argues that combining anti-seizure, anti-inflammatory, neuroprotective, anti-oxidant and anti-growth effects could be a promising anti-epileptogenic strategy in TLE.

Viral vectors are attractive tools to express genes in neurons. Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. However, to which extent Sindbis by itself may affect neurons is not fully understood. We assessed effects of neuronal transduction with a Sindbis viral vector on the transcriptome and proteome in organotypic hippocampal slice cultures, and analyzed the electrophysiological properties of individual CA1 neurons, at 24 h and 72 h after viral vector injection. Whereas Sindbis caused substantial gene expression alterations, changes at the protein level were less pronounced. Alterations in transcriptome and proteome were predominantly limited to proteins involved in mediating anti-viral innate immune responses. Sindbis transduction did not affect the intrinsic electrophysiological properties of individual neurons: the membrane potential and neuronal excitability were similar between transduced and non-transduced CA1 neurons up to 72 h after Sindbis injection. Synaptic currents also remained unchanged upon Sindbis transduction, unless slices were massively infected for 72 h. We conclude that Sindbis viral vectors at low transduction rates are suitable for studying short-term effects of a protein of interest on electrophysiological properties of neurons, but not for studies on the regulation of gene expression.

Hersennetwerken moeten stabiel en adaptief zijn. Dit wordt gereguleerd binnen de individuelen componenten: (inter-)neuronen, en hun organisatie in het netwerk. De dynamische aanpassing van de A-stroom (IA), C-stroom (IC) en H-stroom (Ih) is hier onderzocht, op cel- en netwerkniveau in de hippocampus (CA1) van het muizenbrein. De vraag die centraal stond is wat het effect van zulke schaling op het netwerk en de individuele cellen is en en of hiermee Pyramidaal neuronen (PYR) in Oriens Lacunosum-Moleculare interneuronen (OLM) veranderd kunnen worden? Dit zijn de bevindingen: - OLMs hebben een grote Ih stroom, die bij verhoging ook resonantie voor thetafrequenties veroorzaakte. - Verhoogde synaptische input door thetaburst-stimulatie in een populatie, veranderde excitabiliteit. fEPSP en PS-parameters lieten LTP-inductie zien en intrinsieke schaling gedurende de theta-stimulatie. Ih was cruciaal in de ingewikkelde relatie tussen synaptische en intrinsieke schaling. - Een “Dynamic Clamp” (DC) systeem op een Raspberry Pi werd gepresenteerd, wat via “soft-clamping” zorgde dat experimenten van een van tevoren gedefinieerd membraan potentiaal starten in “current-clamp”. Daarnaast modelleerde het kaliumstromen. - OLMs hebben een grotere nahyperpolarisatie, vuren sneller en raken niet in depolarisatie block vergeleken met PYRs. De verschillen in kalium stromen tussen OLM en PYR konden PYRs het vuurgedrag van OLM geven, behalve het snelle vuren, wat waarschijnlijk verschillen in Na-stromen werd veroorzaakt. Wij concluderen dat OLMs Ih schalen zoals PYRs, dit beïnvloedt de resonantiefrequentie en excitabiliteit wat bijdraagt netwerk stabiliteit en andere netwerk intrinsieke eigenschappen. Kaliumstromen karakteriseren de cel specifieke verschillen in actiepotentialen en dragen zo bij in de organisatie en stabilisatie van een netwerk.

The perirhinal (PER) and lateral entorhinal (LEC) cortex function as a gateway for information transmission between (sub)cortical areas and the hippocampus. It is hypothesized that the amygdala, a key structure in emotion processing, can modulate PER-LEC neuronal activity before information enters the hippocampal memory pathway. This study determined the integration of synaptic activity evoked by simultaneous neocortical and amygdala electrical stimulation in PER-LEC deep layer principal neurons and parvalbumin (PV) interneurons in mouse brain slices. The data revealed that both deep layer PER-LEC principal neurons and PV interneurons receive synaptic input from the neocortical agranular insular cortex (AiP) and the lateral amygdala (LA). Furthermore, simultaneous stimulation of the AiP and LA never reached the firing threshold in principal neurons of the PER-LEC deep layers. PV interneurons however, mainly showed linear summation of simultaneous AiP and LA inputs and reached their firing threshold earlier. This early PV firing was reflected in the forward shift of the evoked inhibitory conductance in principal neurons, thereby creating a more precise temporal window for coincidence detection, which likely plays a crucial role in information processing.

Summary VPS10P domain receptors emerge as central regulators of intracellular protein sorting in neurons with relevance for various brain pathologies. Here, we identified a role for the family member SorCS2 in protection of neurons from oxidative stress and epilepsy-induced cell death. We show that SorCS2 acts as sorting receptor that sustains cell surface expression of the neuronal amino acid transporter EAAT3 to facilitate import of cysteine, required for synthesis of the reactive oxygen species scavenger glutathione. Lack of SorCS2 causes depletion of EAAT3 from the plasma membrane and impairs neuronal cysteine uptake. As a consequence, SorCS2-deficient mice exhibit oxidative brain damage that coincides with enhanced neuronal cell death and increased mortality during epilepsy. Our findings highlight a protective role for SorCS2 in neuronal stress response and provide a possible explanation for upregulation of this receptor seen in surviving neurons of the human epileptic brain., Graphical Abstract, Highlights • SorCS2 is upregulated in neurons of human and mouse epileptic brains • SorCS2 protects from oxidative damage and death in a mouse model of epilepsy • Cysteine transporter EAAT3 is a target for SorCS2-dependent intracellular sorting • SorCS2 loss impairs neuronal cysteine import and reduces glutathione levels, Malik et al. uncover a mechanism crucial for neuronal oxidative stress defense. They show that sorting receptor SorCS2 facilitates cell surface expression of cysteine transporter EAAT3. Loss of Sorcs2 impairs neuronal cysteine import and glutathione production, increases vulnerability to oxidative damage, and aggravates epilepsy-induced brain pathology and death in mice.

The perirhinal (PER) - lateral entorhinal (LEC) network plays a pivotal role in the information transfer between the neocortex and the hippocampus. Anatomical studies have shown that the connectivity is organized bi-directionally: the superficial layers consist of projections running from the neocortex via the PER-LEC network to the hippocampus while the deep layers form the output pathway back to the neocortex. Although these pathways are characterized anatomically, the functional organization of the superficial and deep connections in the PER-LEC network remains to be revealed. We performed paired recordings of superficial and deep layer principal neurons and found that a larger population of superficial neurons responded with action potential firing in response to superficial cortical input, compared to the deep layer population. This suggested that the superficial network can carry information from the cortex towards the hippocampus. The relation between the excitatory and inhibitory input onto the deep and superficial principal neurons showed that the window of net excitability was larger in superficial principal neurons. We performed paired recordings in superficial layer principal neurons and parvalbumin (PV) expressing interneurons to address how this window of opportunity for spiking is affected in superficial principal neurons. The PV interneuron population initiated inhibition at a very consistent timing with increasing stimulus intensity, whereas the excitation temporally shifted to ensure action potential firing. These data indicate that superficial principal neurons can transmit cortical synaptic input through the PER-LEC network because these neurons have a favorable window of opportunity for spiking in contrast to deep neurons.