Reelin differentially shapes dendrite morphology of medial entorhinal cortical ocean and island cells.

The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.

Reelin Regulates Developmental Desynchronization Transition of Neocortical Network Activity.

During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.

Tiagabine and zonisamide differentially regulate the glial properties in an astrocyte-microglia co-culture model of inflammation.

Due to the role of astrocytes and microglia in the pathophysiology of epilepsy and limited studies of antiseizure medication (ASM) effects on glial cells, we studied tiagabine (TGB) and zonisamide (ZNS) in an astrocyte-microglia co-culture model of inflammation. Different concentrations of ZNS (10, 20, 40, 100 µg/ml) or TGB (1, 10, 20, 50 µg/ml) were added to primary rat astrocytes co-cultures with 5-10% (M5, physiological conditions) or 30-40% (M30, pathological inflammatory conditions) microglia for 24 h, aiming to study glial viability, microglial activation, connexin 43 (Cx43) expression and gap-junctional coupling. ZNS led to the reduction of glial viability by only 100 µg/ml under physiological conditions. By contrast, TGB revealed toxic effects with a significant, concentration-dependent reduction of glial viability under physiological and pathological conditions. After the incubation of M30 co-cultures with 20 µg/ml TGB, the microglial activation was significantly decreased and resting microglia slightly increased, suggesting possible anti-inflammatory features of TGB under inflammatory conditions. Otherwise, ZNS caused no significant changes of microglial phenotypes. The gap-junctional coupling was significantly decreased after the incubation of M5 co-cultures with 20 and 50 µg/ml TGB, which can be related to its anti-epileptic activity under noninflammatory conditions. A significant decrease of Cx43 expression and cell-cell coupling was found after the incubation of M30 co-cultures with 10 µg/ml ZNS, suggesting additional anti-seizure effects of ZNS with the disruption of glial gap-junctional communication under inflammatory conditions. TGB and ZNS differentially regulated the glial properties. Developing novel ASMs targeting glial cells may have future potential as an "add-on" therapy to classical ASMs targeting neurons.

The impact of early life maternal deprivation on the perineuronal nets in the prefrontal cortex and hippocampus of young adult rats.

Early life stress negatively impacts brain development and affects structure and function of parvalbumin immunopositive (PV+) inhibitory neurons. Main regulators of PV+ interneurons activity and plasticity are perineuronal nets (PNNs), an extracellular matrix formation that enwraps PV+ interneurons mainly in the neocortex and hippocampus. To experimentally address the impact of early life stress on the PNNs and PV+ interneurons in the medial prefrontal cortex and dorsal hippocampus in rats, we employed a 24 h maternal deprivation protocol. We show that maternal deprivation in the medial prefrontal cortex of adult rats caused a decrease in density of overall PNNs and PNNs that enwrap PV+ interneurons in the rostral cingulate cortex. Furthermore, a staining intensity decrease of overall PNNs and PNN+/PV+ cells was found in the prelimbic cortex. Finally, a decrease in both intensity and density of overall PNNs and PNNs surrounding PV+ cells was observed in the infralimbic cortex, together with increase in the intensity of VGAT inhibitory puncta. Surprisingly, maternal deprivation did not cause any changes in the density of PV+ interneurons in the mPFC, neither had it affected PNNs and PV+ interneurons in the hippocampus. Taken together, our findings indicate that PNNs, specifically the ones enwrapping PV+ interneurons in the medial prefrontal cortex, are affected by early life stress.

Brivaracetam exhibits mild pro-inflammatory features in an in vitro astrocyte-microglia co-culture model of inflammation.

Implications of glia in the pathophysiology of epilepsy raise the question of how these cells besides neurons are responsive to antiseizure medications (ASMs). Understanding ASM effects on glia and glia-mediated inflammation may help to explore astrocytes and microglia as potential targets for alternative anti-epileptogenic therapies. The aim of this study was to investigate the effects of the new generation ASM brivaracetam (BRV) in an astrocyte-microglia co-culture model of inflammation. Primary rat astrocytes co-cultures containing 5%-10% (M5, "physiological" conditions) or 30%-40% (M30, "pathological inflammatory" conditions) of microglia were treated with different concentrations of BRV (0.5, 2, 10, and 20 µg/ml) for 24 h. Glial cell viability was measured by MTT assay. Microglial activation states were analyzed by immunocytochemistry and astroglial connexin 43 (Cx43) expression by Western blot analysis and immunocytochemistry. Gap-junctional coupling was studied via Scrape Loading. Incubation with high, overdose concentration (20 µg/ml) of BRV significantly reduced the glial cell viability under physiological conditions (p < 0.01: **). Treatment with BRV in therapeutic concentrations (0.5 and 2 µg/ml) reduced the resting microglia (p < 0.05: *) and increased the microglial activation under inflammatory conditions (p < 0.01: **). Astroglial Cx43 expression was not affected. The gap-junctional coupling significantly increased only by 0.5 µg/ml BRV under physiological conditions (p < 0.05: *). Our findings suggest mild pro-inflammatory, in vitro features of BRV with regard to microglia morphology. BRV showed no effects on Cx43 expression and only limited effects on gap-junctional coupling. Reduction of glial viability by overdose BRV indicates possible toxic effects.

Tetrodotoxin prevents heat-shock induced granule cell dispersion in hippocampal slice cultures.

Granule cell dispersion (GCD) has been associated as a pathological feature of temporal lobe epilepsy (TLE). Early-life epileptiform activity such as febrile seizures has been proposed to have a causal link to developing chronic TLE. During postnatal development, the hippocampus may be particularly vulnerable to hyperexcitability-induced insults since neuronal migration and differentiation are still ongoing in the hippocampus. Further, the extracellular matrix (ECM), here in particular the protein reelin, has been implicated in the pathophysiology of GCD. Thus, loss of reelin-expressing cells, Cajal-Retzius cells and subsets of interneurons, may be related to GCD. To study the possible role of febrile seizures, we previously induced GCD in vitro by subjecting hippocampal slice cultures to a transient heat-shock, which was not accompanied by loss of Cajal-Retzius cells. In order to examine the mechanisms involved in heat-shock induced GCD, the present study aimed to determine whether such dispersion could be prevented by blocking cellular electrical activity. Here we show that the extent of heat-shock induced GCD could be significantly reduced by treatment with the sodium channel blocker tetrodotoxin (TTX), suggesting that electrical activity is an important factor involved in heat-shock induced GCD.

Considering the Role of Extracellular Matrix Molecules, in Particular Reelin, in Granule Cell Dispersion Related to Temporal Lobe Epilepsy.

The extracellular matrix (ECM) of the nervous system can be considered as a dynamically adaptable compartment between neuronal cells, in particular neurons and glial cells, that participates in physiological functions of the nervous system. It is mainly composed of carbohydrates and proteins that are secreted by the different kinds of cell types found in the nervous system, in particular neurons and glial cells, but also other cell types, such as pericytes of capillaries, ependymocytes and meningeal cells. ECM molecules participate in developmental processes, synaptic plasticity, neurodegeneration and regenerative processes. As an example, the ECM of the hippocampal formation is involved in degenerative and adaptive processes related to epilepsy. The role of various components of the ECM has been explored extensively. In particular, the ECM protein reelin, well known for orchestrating the formation of neuronal layer formation in the cerebral cortex, is also considered as a player involved in the occurrence of postnatal granule cell dispersion (GCD), a morphologically peculiar feature frequently observed in hippocampal tissue from epileptic patients. Possible causes and consequences of GCD have been studied in various in vivo and in vitro models. The present review discusses different interpretations of GCD and different views on the role of ECM protein reelin in the formation of this morphological peculiarity.

Corrigendum: Postnatal Developmental Expression Profile Classifies the Indusium Griseum as a Distinct Subfield of the Hippocampal Formation.

[This corrects the article DOI: 10.3389/fcell.2020.615571.].

Mice lacking perforin have improved regeneration of the injured femoral nerve.

The role that the immune system plays after injury of the peripheral nervous system is still not completely understood. Perforin, a natural killer cell- and T-lymphocyte-derived enzyme that mediates cytotoxicity, plays important roles in autoimmune diseases, infections and central nervous system trauma, such as spinal cord injury. To dissect the roles of this single component of the immune response to injury, we tested regeneration after femoral nerve injury in perforin-deficient (Pfp-/-) and wild-type control mice. Single frame motion analysis showed better motor recovery in Pfp-/- mice compared with control mice at 4 and 8 weeks after injury. Retrograde tracing of the motoneuron axons regrown into the motor nerve branch demonstrated more correctly projecting motoneurons in the spinal cord of Pfp-/- mice compared with wild-types. Myelination of regrown axons measured by g-ratio was more extensive in Pfp-/- than in wild-type mice in the motor branch of the femoral nerve. Pfp-/- mice displayed more cholinergic synaptic terminals around cell bodies of spinal motoneurons after injury than the injured wild-types. We histologically analyzed lymphocyte infiltration 10 days after surgery and found that in Pfp-/- mice the number of lymphocytes in the regenerating nerves was lower than in wild-types, suggesting a closed blood-nerve barrier in Pfp-/- mice. We conclude that perforin restricts motor recovery after femoral nerve injury owing to decreased survival of motoneurons and reduced myelination.

Reelin restricts dendritic growth of interneurons in the neocortex.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.

Corrigendum: Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation.

[This corrects the article DOI: 10.3389/fimmu.2020.624612.].

Heat-Shock Induces Granule Cell Dispersion and Microgliosis in Hippocampal Slice Cultures.

Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as in vitro model to analyze the effect of a transient temperature increase on the dg morphology. We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal-Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.

Impact of Depletion of Microglia/Macrophages on Regeneration after Spinal Cord Injury.

Microglia/macrophages play important functional roles in regeneration after central nervous system injury. Infiltration of circulating macrophages and proliferation of resident microglia occur within minutes following spinal cord injury. Activated microglia/macrophages clear tissue debris, but activation over time may hamper repair. To study the role of these cells in regeneration after spinal cord injury we used CD11b-herpes simplex virus thymidine kinase (HSVTK) (TK) transgenic mice, in which viral thymidine kinase activates ganciclovir toxicity in CD11b-expressing myeloid cells, including macrophages and microglia. A severe reduction in number of these cells was seen in TK versus wild-type littermate mice at 1 week and 5 weeks after injury, and numbers of Mac-2 expressing activated microglia/macrophages were almost completely reduced at these time points. One week after injury TK mice showed better locomotor recovery, but recovery was similar to wild-type mice as measured weekly up to 5 weeks thereafter. At 5 weeks after injury, numbers of axons at the lesion site and neurons in the lumbar spinal cord did not differ between groups. Also, catecholaminergic innervation of spinal motoneurons was similar. However, cholinergic innervation was lower and glial scarring was increased in TK mice compared to wild-type mice. We conclude that reducing numbers of CD11b-expressing cells improves locomotor recovery in the early phase after spinal cord injury, but does not affect recovery in the following 4 weeks. These observations point to differences in outcomes of astrocytic response and cholinergic innervation under CD11b cell ablation, which are, however, not reflected in the locomotor parameters analyzed at 5 weeks after injury.

Reelin signaling modulates GABAB receptor function in the neocortex.

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1.

The expanded HTT CAG repeat causing Huntington's disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans, we performed comprehensive quantitative analyses of CAG expansion in ~50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

Biolistic transfection and expression analysis of acute cortical slices.

BACKGROUND: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 µm) confirm previously published protocols, the amount of 5 µg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION: The method is optimal for short-time investigation of calcium signals in acute slices.

Lateralization of increased density of Iba1-immunopositive microglial cells in the anterior midcingulate cortex of schizophrenia and bipolar disorder.

There is increasing evidence from genetic, biochemical, pharmacological, neuroimaging and post-mortem studies that immunological dysregulation plays a crucial role in the pathogenesis of psychoses. The involvement of microglia in schizophrenia and bipolar disorder (BD) has remained controversial, however, since results from various post-mortem studies are still inconclusive. Here, we analyzed the estimated density of microglia of age-matched individuals with schizophrenia (n = 17), BD (n = 13), and non-psychiatric control subjects (n = 17) in the anterior midcingulate cortex (aMCC), a brain area putatively involved in the pathogenesis of psychoses, using ionized calcium binding adaptor molecule 1 (Iba1)-immunohistochemistry. The microglial cells displayed a homogenously distributed Iba1-staining pattern in the aMCC with slightly varying activation states in all three groups. The estimated microglial densities did not differ significantly between individuals with schizophrenia, BD and control subjects. Remarkably, when both hemispheres were investigated separately within the three groups, the density was significantly lateralized towards the right aMCC in schizophrenia (p = 0.01) and-even more evident-in BD subjects (p = 0.008). This left-right lateralization was not observed in the control group (p = 0.52). Of note, microglial density was significantly lower in BD individuals who did not commit suicide compared with BD individuals who died from suicide (p = 0.002). This difference was not observed between individuals with BD who committed suicide and controls. The results, tentatively interpreted, suggest a hitherto unknown increased lateralization of microglial density to the right hemisphere in both psychiatric groups. If confirmed in independent samples, lateralization should be considered in all post-mortem studies on microglia. Density differences between suicide and non-suicide individuals needs further elucidation.

Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation.

Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.