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Structural epilepsies display complex immune activation signatures; however, it is unclear which neuroinflammatory pathways drive disease pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor β, interferon α/β and nuclear factor erythroid 2-related factor 2 pathways among other neuroinflammatory mechanisms. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack USP15 underwent intrahippocampal kainate injections to induce mTLE and to investigate the impact of USP15 downregulation at the molecular and phenotypic levels. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wildtype littermates. Cell death, reactive gliosis and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, the lack of USP15 did not alter brain inflammation signatures. Likewise, induced deletion of Usp15 in chronic epilepsy neither affected seizure generation, nor cell death, gliosis or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in mTLE and has no impact on epileptic activity in a mouse model. Although previous reports implicated a checkpoint function for USP15 in inflammation, our results do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

Hippocampal sclerosis (HS) is often associated with gray-white matter blurring (GMB) of the anterior temporal lobe. In this study, twenty patients with unilateral temporal lobe epilepsy and HS were studied with 3 T MRI including T1 MP2RAGE and DTI/DMI sequences. Anterior temporal lobe white matter T1 relaxation times and diffusion measures were analyzed on the HS side, on the contralateral side, and in 10 normal controls. Resected brain tissue of three patients without GMB and four patients with GMB was evaluated ultrastructurally regarding axon density and diameter, the relation of the axon diameter to the total fiber diameter (G-ratio), and the thickness of the myelin sheath. Hippocampal sclerosis GMB of the anterior temporal lobe was related to prolonged T1 relaxation and axonal loss. A less pronounced reduction in axonal fraction was also found on imaging in GMB-negative temporal poles compared with normal controls. Contralateral values did not differ significantly between patients and normal controls. Reduced axonal density and axonal diameter were histopathologically confirmed in the temporopolar white matter with GMB compared to temporal poles without. These results confirm that GMB can be considered an imaging correlate for disturbed axonal maturation that can be quantified with advanced diffusion imaging.

Hippocampal sclerosis (HS) in Temporal Lobe Epilepsy (TLE) shows neuronal death in cornu ammonis (CA)1, CA3, and CA4. It is known that granule cells and CA2 neurons survive and their axons, the mossy fibers (MF), lose their target cells in CA3 and CA4 and sprout to the granule cell layer and molecular layer. We examined in TLE patients and in a mouse epilepsy model, whether MF sprouting is directed to the dentate gyrus or extends to distant CA regions and whether sprouting is associated with death of target neurons in CA3 and CA4. In 319 TLE patients, HS was evaluated by Wyler grade and International League against Epilepsy (ILAE) types using immunohistochemistry against neuronal nuclei (NeuN). Synaptoporin was used to colocalize MF. In addition, transgenic Thy1-eGFP mice were intrahippocampally injected with kainate and sprouting of eGFP-positive MFs was analyzed together with immunocytochemistry for regulator of G-protein signaling 14 (RGS14). In human HS Wyler III and IV as well as in ILAE 1, 2, and 3 specimens, we found synaptoporin-positive axon terminals in CA2 and even in CA1, associated with the extent of granule cell dispersion. Sprouting was seen in cases with cell death of target neurons in CA3 and CA4 (classical severe HS ILAE type 1) but also without this cell death (atypical HS ILAE type 2). Similarly, in epileptic mice eGFP-positive MFs sprouted to CA2 and beyond. The presence of MF terminals in the CA2 pyramidal cell layer and in CA1 was also correlated with the extent of granule cell dispersion. The similarity of our findings in human specimens and in the mouse model highlights the importance and opens up new chances of using translational approaches to determine mechanisms underlying TLE.

Local extinction and undetected presence are two very different biological phenomena, but they can be challenging to differentiate. Stochastic environments hamper the development of standardized monitoring schemes for wildlife, and make it more challenging to plan and evaluate the success of conservation efforts. To avoid reintroductions of species at risk that could jeopardize extant populations, managers attempting translocation events require a higher level of confidence that a failure to confirm presence represents a true absence. For many pond breeding amphibians, monitoring of the breeding population occurs indirectly through larval surveys. Larval development and successful recruitment only occurs after a sequence of appropriate environmental conditions, thus it is possible for a breeding population of adults to exist at a site but for detectability of the species to be functionally zero. We investigate how annual variability in detection influences long-term monitoring efforts of Reticulated Flatwoods Salamanders (Ambystoma bishopi) breeding in 29 wetlands in Florida. Using 8 years of historic dip net data, we simulate plausible monitoring scenarios that incorporate environmental stochasticity into estimates of detection probability. We found that annual variation in environmental conditions precluded a high degree of certainty in predicting site status for low-intensity monitoring schemes. Uncertainty was partly alleviated by increasing survey effort, but even at the highest level of sampling intensity assessed, multiple years of monitoring are required to confidently determine presence/absence at a site. Combined with assessments of habitat quality and landscape connectivity, our results can be used to identify sites suitable for reintroduction efforts. Our methodologies can be generally applied to increase the effectiveness of surveys for diverse organisms for which annual variability in detectability is known.

The performance of available optogenetic inhibitors remains insufficient due to low light sensitivity, short-lasting photocurrents, and unintended changes in ion distributions. To overcome these limitations, a novel potassium channel-based optogenetic silencer was developed and successfully applied in various in vitro and acute in vivo settings (Bernal Sierra et al., 2018). This tool, a two-component construct called PACK, comprises a photoactivated adenylyl cyclase (bPAC) and a cAMP-dependent potassium channel (SthK). Here, we examined the long-term inhibitory action and side effects of the PACK construct in healthy and epileptic adult male mice. We targeted hippocampal CA1 pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from the CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-minute intermittent illumination, especially in the beta and gamma frequency ranges. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Additionally, light-activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. In chronically epileptic mice, the dark activity of bPAC/PACK in CA1 prevented the spread of spontaneous epileptiform activity from the seizure focus to the contralateral bPAC/PACK-expressing hippocampus. Taken together, the PACK tool is a potent optogenetic inhibitor but requires refinement of its light-sensitive domain to avoid unexpected physiological changes.Significance statementOptogenetics allows precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Unfortunately, available optogenetic inhibitors are not suitable for prolonged use. The newly developed two-component potassium channel-based optogenetic inhibitor, PACK, has been identified as a potent silencer of neurons in various acute experiments. Here, we characterized the PACK construct in freely behaving healthy and epileptic mice. We targeted the PACK silencer specifically to CA1 pyramidal neurons, where illumination with short light pulses at low frequencies reliably reduced neuronal activity. In chronically epileptic mice, PACK prevented the spread of epileptiform activity from the seizure focus to the contralateral PACK-expressing hippocampus. The major disadvantage of the PACK silencer is its light-sensitive domain, the bPAC adenylyl cyclase, which may induce side effects.

American Shad (Alosa sapidissima), Hickory Shad (A. mediocris), and river herrings (Alewife A. pseudoharengus and Blueback Herring A. aestivalis) are anadromous pelagic fishes, which as adults spend most of the annual cycle at sea, but enter the coastal rivers in spring to spawn. Once as one of the most valuable fisheries along the Atlantic coast, Alosa populations have declined in recent decades and current populations are at historic lows. Various management actions have been conducted to restore the populations, and stocks in different river systems display different demographic trends. Demonstration of synthetic diagnostics on the factors impacting these populations is important to better conserve this species group. We developed a Bayesian hierarchical spatiotemporal model to identify the population trends of these species among rivers in the Chesapeake Bay based on results of surveys conducted by the Virginia Department of Game and Inland Fisheries and Maryland Department of Natural Resources and to identify environmental and anthropogenic factors influencing their distribution and abundance. The hierarchical model structure helped to diagnose river-specific population trends and impacts of surrounding factors, and decrease uncertainties in rivers with less samples available. The results demonstrate river-specific heterogeneity of spatiotemporal dynamics of these species and indicate river-specific impacts of multiple factors, including water temperature, river flow, chlorophyll a concentration, and total phosphorus concentration, on their population dynamics. Atlantic Multidecadal Oscillation and Gulf Stream meanders displayed significant influence on the inter-annual trends of Alosa species in rivers with more data available. The results would help to develop river- and species-specific management strategies to recover these species. Virginia Tech; Virginia Department of Game and Inland Fisheries; Maryland Department of Natural Resources; Atlantic States Marine Fisheries Commission Published version We acknowledge supporters, including Virginia Tech, Virginia Department of Game and Inland Fisheries, Maryland Department of Natural Resources, and Atlantic States Marine Fisheries Commission. We would like to thank Dr. Can Zhou for his help on model development. We also acknowledge Advanced Research Computing at Virginia Tech () for providing computational resources and technical support that have contributed to the results reported within this paper. Attention to the comments of the associate editor and anonymous reviewers led to strengthening of the manuscript.

Long term implantation of (micro-)probes into neural tissue cause unique and disruptive responses to these foreign bodies. In this study, we present the transcriptional trajectory of glial cells responding to chronic implantation of flexible micro-probes for up to 18 weeks. Transcriptome analysis shows a rapid activation of microglial cells and a strong upregulation of reactive astrocytic genes, which is lost over the full duration of the implant period. Most interestingly, animals that were implanted for 18 weeks show a transcriptional profile similar to non-implanted controls, with increased expression of genes associated with wound healing and angiogenesis, which raises hope of a normalization of the neuropil to the pre-injury state when using flexible probes. Nevertheless, our data show, that a subset of genes upregulated after 18 weeks belong to the family of immediate early genes, which would indicate that structural and functional remodeling has not been completed at this time point. Our results confirm and extend previous work on the molecular changes resulting from the presence of intraneural probes and provide a rational basis for developing intervention strategies to control them.

Dendritic spines are highly dynamic neuronal compartments that control the synaptic transmission between neurons. Spines form ultrastructural units, coupling synaptic contact sites to the dendritic shaft and often harbor a spine apparatus organelle, composed of smooth endoplasmic reticulum, which is responsible for calcium sequestration and release into the spine head and neck. The spine apparatus has recently been linked to synaptic plasticity in adult human cortical neurons. While the morphological heterogeneity of spines and their intracellular organization has been extensively demonstrated in animal models, the influence of spine apparatus organelles on critical signaling pathways, such as calcium-mediated dynamics, is less well known in human dendritic spines. In this study we used serial transmission electron microscopy to anatomically reconstruct nine human cortical spines in detail as a basis for modeling and simulation of the calcium dynamics between spine and dendrite. The anatomical study of reconstructed human dendritic spines revealed that the size of the postsynaptic density correlates with spine head volume and that the spine apparatus volume is proportional to the spine volume. Using a newly developed simulation pipeline, we have linked these findings to spine-to-dendrite calcium communication. While the absence of a spine apparatus, or the presence of a purely passive spine apparatus did not enable any of the reconstructed spines to relay a calcium signal to the dendritic shaft, the calcium-induced calcium release from this intracellular organelle allowed for finely tuned “all-or-nothing” spine-to-dendrite calcium coupling; controlled by spine morphology, neck plasticity, and ryanodine receptors. Our results suggest that spine apparatus organelles are strategically positioned in the neck of human dendritic spines and demonstrate their potential relevance to the maintenance and regulation of spine-to-dendrite calcium communication.

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal epilepsy in adults and is typically associated with hippocampal sclerosis and drug-resistant seizures. As an alternative to curative epilepsy surgery, brain stimulation evolves as a promising approach for seizure-interference. However, particularly in MTLE with severe hippocampal sclerosis, current stimulation protocols are often not effective. Here, we show that optogenetic low-frequency stimulation (oLFS) of entorhinal afferents exhibits unprecedented anti-ictogenic effects in chronically epileptic mice. Photostimulation at 1 Hz resulted in an almost complete suppression of focal seizures, independent of the degree of hippocampal sclerosis. Furthermore, by performing oLFS for 30 min before a pro-convulsive stimulus, seizure generalization was successfully prevented. Finally, acute slice experiments revealed a decreased excitability upon oLFS, which may partially explain the observed anti-epileptic effects. Taken together, our results suggest that oLFS of entorhinal afferents constitutes a promising approach for seizure control in MTLE.

Hippocampal Low-Frequency Stimulation Prevents Seizure Generation in a Mouse Model of Mesial Temporal Lobe Epilepsy Paschen E, Elgueta C, Heining K, et al. Elife. 2020;9:e54518. doi:10.7554/eLife.54518Mesial temporal lobe epilepsy (MTLE) is the most common form of focal, pharmacoresistant epilepsy in adults and is often associated with hippocampal sclerosis. Here, we established the efficacy of optogenetic and electrical low-frequency stimulation (LFS) in interfering with seizure generation in a mouse model of MTLE. Specifically, we applied LFS in the sclerotic hippocampus to study the effects on spontaneous subclinical and evoked generalized seizures. We found that stimulation at 1 Hz for 1 hour resulted in an almost complete suppression of spontaneous seizures in both hippocampi. This seizure-suppressive action during daily stimulation remained stable over several weeks. Furthermore, LFS for 30 minutes before a proconvulsive stimulus successfully prevented seizure generalization. Finally, acute slice experiments revealed a reduced efficacy of perforant path transmission onto granule cells upon LFS. Taken together, our results suggest that hippocampal LFS constitutes a promising approach for seizure control in MTLE.

Objectives Focal cortical dysplasias (FCDs) are local malformations of the human neocortex and a leading cause of medically intractable epilepsy. FCDs are characterized by local architectural disturbances of the neocortex and often by a blurred gray-white matter boundary indicating abnormal white matter myelination. We have recently shown that myelination is also compromised in the gray matter of dysplastic areas, since transcripts encoding factors for oligodendrocyte differentiation and myelination are downregulated and myelin fibers appear fractured and disorganized. Methods Here, we characterized the gray matter-associated myelination pathology in detail by in situ hybridization, immunohistochemistry, and electron microscopy with markers for myelin, mature oligodendrocytes, and oligodendrocyte precursor cells in tissue sections of FCD IIa and control cortices. In addition, we isolated oligodendrocyte precursor cells from resected dysplastic tissue and performed proliferation assays. Results We show that the proportion of myelinated gray matter is similar in the dysplastic cortex to that in controls and myelinated fibers extend up to layer III. On the ultrastructural level, however, we found that the myelin sheaths of layer V axons are thinner in dysplastic specimens than in controls. In addition, the density of oligodendrocyte precursor cells and of mature oligodendrocytes was reduced. Finally, we show for the first time that oligodendrocyte precursor cells isolated from resected dysplastic cortex have a reduced proliferation capacity in comparison to controls. Significance These results indicate that proliferation and differentiation of oligodendrocyte precursor cells and the formation of myelin sheaths are compromised in FCD and might contribute to the epileptogenicity of this cortical malformation.

We present a joint effort at Virginia Tech between a research group in the Department of Fish and Wildlife Conservation and Data Services in the University Libraries to improve data management for long-term ecological field research projects in the Florida Panhandle. Consultative research data management support from Data Services in the University Libraries played an integral role in development of the training curriculum. Emphasizing the importance of data quality to the field workers at the beginning of this training curriculum was a vital part of its success. Also critical for success was the research group’s investment of time and effort to work with field workers and improve data management systems. We compare this case study to three others in the literature to compare and contrast data management processes and procedures. This case study serves as one example of how targeted training and efforts in data and project management for a research project can lead to substantial improvements in research data quality. Preprint This article has been accepted for publication; this is the preprint (i.e. article prior to publication).

Objective Focal cortical dysplasia (FCD) is a major cause of pharmacoresistant focal epilepsy. Little is known about the pathomechanisms underlying the characteristic cytoarchitectural abnormalities associated with FCD. In the present study, a broad panel of markers identifying layer-specific neuron subpopulations was applied to characterize dyslamination and structural alterations in FCD with balloon cells (FCD 2b). Methods Pan-neuronal neuronal nuclei (NeuN) and layer-specific protein expression (Reelin, Calbindin, Calretinin, SMI32 (nonphosphorylated neurofilament H), Parvalbumin, transducin-like enhancer protein 4 (TLE4), and Vimentin) was studied by immunohistochemistry on paraffin sections of FCD2b cases (n = 22) and was compared to two control groups with (n = 7) or without epilepsy (n = 4 postmortem cases). Total and layer-specific neuron densities were systematically quantified by cell counting considering age at surgery and brain region. Results We show that in FCD2b total neuron densities across all six cortical layers were not significantly different from controls. In addition, we present evidence that a basic laminar arrangement of layer-specific neuron subtypes was preserved despite the severe disturbance of cortical structure. SMI32-positive pyramidal neurons showed no significant difference in total numbers, but a reduction in layers III and V. The densities of supragranular Calbindin- and Calretinin-positive interneurons in layers II and III were not different from controls, whereas Parvalbumin-expressing interneurons, primarily located in layer IV, were significantly reduced in numbers when compared to control cases without epilepsy. In layer VI, the density of TLE4-positive projection neurons was significantly increased. Altogether, these data show that changes in cellular composition mainly affect deep cortical layers in FCD2b. Significance The application of a broad panel of markers defining layer-specific neuronal subpopulations revealed that in FCD2b neuronal diversity and a basic laminar arrangement are maintained despite the severe disturbance of cytoarchitecture. Moreover, it showed that Parvalbumin-positive, inhibitory interneurons are highly vulnerable in contrast to other interneuron subtypes, possibly related to the epileptic condition.

Feral swine Sus scrofa have been implicated as a major threat to sensitive habitats and ecosystems as well as threatened wildlife. Nevertheless, direct and indirect impacts on threatened species (especially small, fossorial species) are not well documented. The decline of the U.S. federally endangered reticulated flatwoods salamander Ambystoma bishopi, categorized as Vulnerable on the IUCN Red List, has been rapid and there are few remaining breeding locations for this species. The flatwoods salamander depends on complex herbaceous vegetation in all life stages, including eggs, larvae and adults. Historically sets of hog tracks have been observed only occasionally in the vicinity of monitored reticulated flatwoods salamander breeding wetlands, and damage to the wetlands had never been recorded. However, during the autumn-winter breeding season of 2013-2014 we observed a large increase in hog sign, including extensive rooting damage, in known flatwoods salamander breeding wetlands. Our objective was to assess the amount of hog sign and damage in these wetlands and to take corrective management actions to curb additional impacts. Of 28 wetlands surveyed for hog sign, presence was recorded at 68%, and damage at 54%. Of the 11 sites known to be occupied by flatwoods salamanders in 2013-2014, 64% had presence, and 55% had damage. We found that regular monitoring of disturbance in wetland habitats was a valuable tool to determine when intervention was needed and to assess the effectiveness of intervention. Habitat damage caused by feral hogs poses a potentially serious threat to the salamanders, which needs to be mitigated using methods to control and exclude hogs from this sensitive habitat. Natural Resources Branch (Jackson Guard) of Eglin Air Force Base; Department of Fisheries and Wildlife Sciences at Virginia Tech We thank the Natural Resources Branch (Jackson Guard) of Eglin Air Force Base and the Department of Fisheries and Wildlife Sciences at Virginia Tech for their financial and logistical support. We specifically thank the leadership and supporting staff at Jackson Guard for their rapid response, Sandy Pizzolato for coordinating fence installation, and the U.S. Fish and Wildlife Service Panama City Ecological Services team for permitting support. Public domain – authored by a U.S. government employee

Visual Abstract, Hypersynchronous network activity is the defining hallmark of epilepsy and manifests in a wide spectrum of phenomena, of which electrographic activity during seizures is only one extreme. The aim of this study was to differentiate between different types of epileptiform activity (EA) patterns and investigate their temporal succession and interactions. We analyzed local field potentials (LFPs) from freely behaving male mice that had received an intrahippocampal kainate injection to model mesial temporal lobe epilepsy (MTLE). Epileptiform spikes occurred in distinct bursts. Using machine learning, we derived a scale reflecting the spike load of bursts and three main burst categories that we labeled high-load, medium-load, and low-load bursts. We found that bursts of these categories were non-randomly distributed in time. High-load bursts formed clusters and were typically surrounded by transition phases with increased rates of medium-load and low-load bursts. In apparent contradiction to this, increased rates of low-load bursts were also associated with longer background phases, i.e., periods lacking high-load bursting. Furthermore, the rate of low-load bursts was more strongly correlated with the duration of background phases than the overall rate of epileptiform spikes. Our findings are consistent with the hypothesis that low-level EA could promote network stability but could also participate in transitions towards major epileptiform events, depending on the current state of the network.

Objective Hippocampal sclerosis is a hallmark of mesial temporal lobe epilepsy (MTLE), comprising gliosis and neuronal loss in the hippocampus. However, dentate granule cells and CA2 pyramidal cells (PCs) survive, as they share physiological characteristics that may render them less sensitive to hyperexcitation in MTLE. Here, we asked whether both engage similar molecular plasticity mechanisms to support their resilience in MTLE. We chose brain-derived neurotrophic factor (BDNF), correlated the expression with activity, and used neuropeptide Y (NPY) and principal cell dispersion as plasticity readout. Methods Adult male mice received a unilateral intrahippocampal kainate injection to induce status epilepticus (SE) and bilateral electrodes into the dentate gyrus and CA2 for in vivo recordings and quantification of epileptiform activity. To assess the time course of Bdnf mRNA expression in these regions, we performed fluorescence in situ hybridization, complemented by immunohistochemistry for NPY and quantification of principal cell dispersion. Results We show that Bdnf expression was transiently up-regulated during SE in the granule cell layer (GCL) and CA2 and, after a slight reduction at 2 days, increased persistently in both regions ipsilaterally. Intrahippocampal recordings revealed a threshold for the duration of SE to induce these changes. Recurrent epileptiform activity developed in the ipsilateral dentate gyrus and CA2 over time and was correlated with Bdnf mRNA levels, although more pronounced in the dentate gyrus. The dispersion of the GCL and CA2 correlated with Bdnf mRNA expression. NPY protein expression was only increased in granule cells and mossy fibers, remaining unchanged in CA2. Significance Our study reveals differential molecular plasticity changes in granule cells and CA2 PCs despite many similarities (epileptiform activity, somatic mossy fiber input, dispersion). These findings contribute to the understanding of common as well as individual characteristics of the cell populations underlying the epileptic hippocampal network.

In a longitudinal mouse study we evaluated whether diffusion tensor imaging (DTI) can monitor microstructural changes after administration of the neuromodulating drug EPO and whether erythropoietin (EPO) has an effect on cognitive performance. Twelve mice (2 groups with 6 mice each) were scanned in a 7T Bruker Biospin animal scanner with a highly resolved DTI sequence before and 16 days after intraperitoneal injections of EPO or saline. All mice underwent behavioral testing (Morris water maze) and histologic evaluation of hippocampal and corpus callosum cell proliferation and oligodendrogenesis. Whole brain DTI analysis showed significant Trace, RD and AD decrease within the dentate gyrus, subiculum, primary motor, somatosensory, and supplementary somatosensory areas and FA increase in the hippocampus, corpus callosum, and fimbria fornix in EPO treated mice only. ROI-based DTI analysis showed significant Trace and RD decrease and FA increase only in the corpus callosum of EPO treated mice, whereas in the dentate gyrus significant Trace, RD, and AD decrease occurred in both, EPO- and control-group. Behavioral tests showed that EPO treated mice performed better and learned faster than controls. Histologically, the number of BrdU-positive nuclei and optical density of DCX-labeled juvenile neurons significantly increased within the dentate gyrus, corpus callosum and fimbria fornix and the number of NG2-positive oligodendrocyte progenitors in corpus callosum and fimbria fornix, respectively. In conclusion we were able to monitor microstructural changes with DTI and showed EPO treatment-related alterations correlating with enhanced dentate gyrus and corpus callosum cell proliferation and better learning capabilities.

See Contreras and Hippenmeyer (doi:10.1093/brain/awy218) for a scientific commentary on this article. Autism spectrum disorders (ASDs) are complex conditions with diverse aetiologies. Szczurkowska et al. demonstrate that two ASD-related molecules – FGFR2 and Negr1 – physically interact to act on the same downstream pathway, and regulate cortical development and ASD-relevant behaviours in mice. Identifying common mechanisms in ASDs may reveal targets for pharmacological intervention., Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases and associated downstream signalling have been strongly implicated in both neurodevelopment and autism spectrum disorders. We found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) similarly affects neuronal migration and spine density during mouse cortical development in vivo and results in impaired core behaviours related to autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signalling by decreasing FGFR2 degradation from the plasma membrane. Accordingly, FGFR2 overexpression rescues all defects due to Negr1 knockdown in vivo. Negr1 knockout mice present phenotypes similar to Negr1-downregulated animals. These data indicate that NEGR1 and FGFR2 cooperatively regulate cortical development and suggest a role for defective NEGR1-FGFR2 complex and convergent downstream ERK and AKT signalling in autism spectrum disorders.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Mesial temporal lobe epilepsy (mTLE) is the most common focal epilepsy in adults and is often refractory to medication. So far, resection of the epileptogenic focus represents the only curative therapy. It is unknown whether pathological processes preceding epilepsy onset are indicators of later disease severity. Using longitudinal multi-modal MRI, we monitored hippocampal injury and tissue reorganization during epileptogenesis in a mouse mTLE model. The prognostic value of MRI biomarkers was assessed by retrospective correlations with pathological hallmarks Here, we show for the first time that the extent of early hippocampal neurodegeneration and progressive microstructural changes in the dentate gyrus translate to the severity of hippocampal sclerosis and seizure burden in chronic epilepsy. Moreover, we demonstrate that structural MRI biomarkers reflect the extent of sclerosis in human hippocampi. Our findings may allow an early prognosis of disease severity in mTLE before its first clinical manifestations, thus expanding the therapeutic window. DOI: http://dx.doi.org/10.7554/eLife.25742.001, eLife digest Roughly one percent of people in the world suffer from epilepsy, a disorder in which individuals experience seizures due to abnormal electrical activity in the brain. Seizures can vary from brief episodes of amnesia or déjà-vu to convulsions and loss of consciousness. In adults, the most common form of epilepsy is known as temporal lobe epilepsy. As the name suggests, this type of epilepsy originates in a region of the brain called the temporal lobe, usually within a structure called the hippocampus. Many patients who develop temporal lobe epilepsy will have experienced a head injury or infection earlier in life that damaged their hippocampus. However, damage to the hippocampus does not always lead to epilepsy. Moreover, many years may pass between the damage and the onset of regular seizures. While some patients find that anti-epileptic drugs can control their seizures, others experience no benefit. For these patients, the only effective treatment is to remove the damaged brain tissue. At present, there is no way of knowing which patients with a damaged hippocampus will go on to develop temporal lobe epilepsy. To identify the deciding factors, Janz, Schwaderlapp, Heining et al. treated mice with a toxin that can damage the hippocampus. After roughly two weeks most of the mice were experiencing regular seizures. Imaging the animals’ brains during this two week period revealed that mice whose hippocampi showed more severe cell death shortly after exposure to the toxin surprisingly developed a milder form of epilepsy. The same was also true for animals whose hippocampi showed signs of being extensively reorganized. Further experiments show that samples of hippocampal tissue from the brains of human patients with temporal lobe epilepsy also showed these same cellular features. The next step is to test whether these changes can be used to predict which patients with hippocampal damage will develop epilepsy later in life. Identifying at-risk individuals would allow them to be treated earlier and hopefully prevent them from developing epilepsy in the first place. DOI: http://dx.doi.org/10.7554/eLife.25742.002

The prevalence of studies that discern the drivers of animal migrations is increasing exponentially, in line with raised concerns over climate-driven shifts in phenological patterns. Amphibians that breed in ephemeral wetlands are particularly vulnerable owing to the stochastic nature of their breeding habitat and high survival and reproductive costs associated with untimely migrations. Knowledge of the effects of climatic cues on the timing of amphibian migration and breeding phenologies is required to understand the potential consequences of climate change and to inform conservation plans for at-risk species. Here we use six years of count data from Eglin Air Force Base, Florida, U.S., collected between 2010 and 2016, to model the phenology of migrations of Ambystoma bishopi. Models were fit with a negative binomial error distribution, with daily values of temperature and precipitation as covariates. Models were compared in an AIC framework. We found that salamander movement was strongly tied to environmental cues. A proclivity to move over a medial range of temperatures and precipitation resulted in a ‘Goldilocks’ set of conditions that facilitate migration. We also discovered a role for endogenous drivers of movement, with day of year predicting above-ground activity regardless of environmental conditions. These results contribute to our basic knowledge of the behavior of A. bishopi and provide managers with useful metrics to maximize the efficacy of conservation actions. More broadly, the methods employed within have utility to amphibian researchers to help predict the effects of climate change and tailor management strategies accordingly.

Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer (GCL) in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSC) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate towards the hilar region where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons.

Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within seven days of cultivation. Accordingly, RT-qPCR analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor (CNTF) accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.

The hippocampus is reciprocally connected with the entorhinal cortex. Although several studies emphasized a role for the entorhinal cortex in mesial temporal lobe epilepsy (MTLE), it remains uncertain whether its synaptic connections with the hippocampus are altered. To address this question, we traced hippocampo-entorhinal and entorhino-hippocampal projections, assessed their connectivity with the respective target cells and examined functional alterations in a mouse model for MTLE. We show that hippocampal afferents to the dorsal entorhinal cortex are lost in the epileptic hippocampus. Conversely, entorhino-dentate projections via the medial perforant path (MPP) are preserved, but appear substantially altered on the synaptic level. Confocal imaging and 3D-reconstruction revealed that new putative contacts are established between MPP fibers and dentate granule cells (DGCs). Immunohistochemical identification of pre- and postsynaptic elements indicated that these contacts are functionally mature synapses. On the ultrastructural level, pre- and postsynaptic compartments of MPP synapses were strongly enlarged. The length and complexity of postsynaptic densities were also increased pointing to long-term potentiation-related morphogenesis. Finally, whole-cell recordings of DGCs revealed an enhancement of evoked excitatory postsynaptic currents. In conclusion, the synaptic rearrangement of excitatory inputs to DGCs from the medial entorhinal cortex may contribute to the epileptogenic circuitry in MTLE.

SUMMARY Purpose: Temporal lobe epilepsy is often accompanied by neuron loss and rewiring in the hippocampus. We hypothesized that the interaction of subnetworks of the entorhinal–hippocampal loop between epileptic events should show significant signatures of these pathologic changes. Methods: We combined simultaneous recording of local field potentials in entorhinal cortex (EC) and dentate gyrus (DG) in freely behaving kainate-injected mice with histologic analyses and computational modeling. Key Findings: In healthy mice, theta band activity was synchronized between EC and DG. In contrast, in epileptic mice, theta activity in the EC was delayed with respect to the DG. A computational neural mass model suggests that hippocampal cell loss imbalances the coupling of subnetworks, introducing the shift. Significance: We show that pathologic dynamics in epilepsy encompass ongoing activity in the entorhinal-hippocampal loop beyond acute epileptiform activity. This predominantly affects theta band activity, which links this shift in entorhinal-hippocampal interaction to behavioral aspects in epilepsy.

Quantifying and comparing habitat selection of related, syntopic species may help elucidate how species partition resources and compete. The Florida Bog Frog (Lithobates okaloosae) is endemic to northwestern Florida and is syntopic with the more widely distributed Bronze Frog (Lithobates clamitans clamitans). Our objective was to determine whether these closely related frogs selected different microhabitat characteristics at male calling sites, which in turn may influence successful reproduction or survival. From 2006 to 2008, we quantified microhabitat characteristics of male calling sites used by both species on Eglin Air Force Base, Florida. We created a suite of a priori models and used paired logistic regression to assess each species' habitat selection. Further, we compared habitat characteristics from each species' most highly supported model to directly compare habitat use. Model selection indicated that calling sites for L. okaloosae were best described by habitat features related to mic...

Environmental temperatures affect nearly all aspects of ectotherm physiology, including terrestrial salamanders. Therefore, habitat disturbances that alter temperature regimes may interact with physiological processes to affect energy budgets of salamanders or constrain surface activity and possibly lead to changes in population-level parameters. We hypothesized that warmer surface temperatures following harvesting of canopy trees could cause surface-active salamanders to expend more energy for metabolism, potentially leaving a smaller proportion of the energy budget available for reproduction or storage. From 2006 to 2008, we quantified temperature regimes of salamander refugia in a field experiment replicated at 4 sites that included plots subjected to a timber harvest and plots not manipulated during this time period. At each site, we quantified temperature regimes in regenerating forest stands which, approximately 10 years earlier, experienced a range of harvest intensity from shelterwood to silvicultural clearcut. Further, we compared energetic parameters including 1) calories required to maintain homeostasis across an active season, 2) abundance of available potential energy (i.e., invertebrate prey), and 3) a measure of growth and storage (i.e., body condition index) among silvicultural treatments for surface-active salamanders. For surface-active eastern red-backed salamanders (Plethodon cinereus), mean calories required for maintenance were approxi- mately 33% greater in recently harvested forest compared to unharvested controls, but body condition was inconsistent among treatments, and invertebrate abundances were similar among treatments but differed by study site. In contrast, we did not detect a treatment effect in any energetic metric 8-14 growing seasons after harvesting. Given that surface-active salamanders in recently harvested forest may be forced to restrain behaviors associated with foraging and mating or trade-off growth or reproduction for increased basic maintenance costs, energetics may be an important but overlooked short-term contributor to observed changes in abundances, reproductive demography, or surface activities after timber harvesting. Managing for both the rapid recovery of understory vegetation and retention of large stumps and logs may help mitigate warming of microclimate for salamanders and should be considered further. 2011 The Wildlife Society.

Conservation of terrestrial salamanders requires consideration for landscape-level features and processes; and, therefore, tools are needed to support planning at large spatial scales. We assessed terrestrial salamander distribution patterns using an existing salamander data set and two previously developed landscape-level spatial tools, a landscape classification (LC) model and flow accumulation (FLOW) model, both based on 100-m2 pixels. The LC model assigned each pixel to one of eight categories defined by slope, aspect and landform. The FLOW model assigned each pixel to one of five categories based on the number of 100-m2 cells that drained into a given cell. Salamander data had been collected in the Allegheny Mountains of West Virginia during 2001–2003 on 543 100-m2 plots located along random transects. Using area-constrained searches we collected 1879 individuals of eleven species. Plethodon cinereus, Desmognathus ochrophaeus, D. monticola and D. fuscus represented 92.8% of all captures. All...

SUMMARY Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate-injected hippocampus, there was an 80% and 85% reduction of GluR2/3- and GluR2/3/calretinin-positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3-, but only a 20% loss of GluR2/3calretinin-immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed.

It is a matter of ongoing debate whether newly generated granule cells contribute to epileptic activity in the hippocampus. To address this question, we investigated neurogenesis and epileptiform activity (EA) along the hippocampal septotemporal axis in the intrahippocampal kainate (KA) mouse model for temporal lobe epilepsy. Multisite intrahippocampal in vivo recordings and immunolabeling for c-Fos showed that the KA-induced status epilepticus (SE) extended along the septotemporal axis of both hippocampi with stronger intensity at ipsilateral temporal and contralateral sites. Accordingly, we found a position-dependent increase in proliferation (incorporation of bromodeoxyuridine) and neurogenesis (immunolabeling for doublecortin): Both were selectively increased in the ipsilateral temporal and entire contralateral subgranular zone, sparing the septal region close to the injection site. The newborn neurons were hyperexcitable and functionally integrated into the hippocampal network as revealed by patch-clamp recordings. Analysis of chronic EA also showed a differential intensity pattern along the hippocampal axis: EA was low in the septal portion with prominent sclerosis and granule cell dispersion but most pronounced in the transition zone where neurogenesis reappeared. In conclusion, SE stimulates neurogenesis in a position-dependent manner and coincidence of neurogenesis and stronger EA distal to the injection site suggests a proepileptogenic effect of increased neurogenesis.

International audience; Mesio-temporal lobe epilepsy (MTLE), the most common drug-resistant epilepsy syndrome, is characterized by the recurrence of spontaneous focal seizures after a latent period that follows, in most patients, an initial insult during early childhood. Many of the mechanisms that have been associated with the pathophysiology of MTLE are known to be regulated by brain-derived neurotrophic factor (BDNF) in the healthy brain and an excess of this neurotrophin could therefore play a critical role in MTLE development. However, such a function remains controversial as other studies revealed that BDNF could, on the contrary, exert protective effects regarding epilepsy development. In the present study, we further addressed the role of increased BDNF/TrkB signaling on the progressive development of hippocampal seizures in the mouse model of MTLE obtained by intrahippocampal injection of kainate. We show that hippocampal seizures progressively developed in the injected hippocampus during the first two weeks following kainate treatment, within the same time-frame as a long-lasting and significant increase of BDNF expression in dentate granule cells. To determine whether such a BDNF increase could influence hippocampal epileptogenesis via its TrkB receptors, we examined the consequences of (i) increased or (ii) decreased TrkB signaling on epileptogenesis, in transgenic mice overexpressing the (i) TrkB full-length or (ii) truncated TrkB-T1 receptors of BDNF. Epileptogenesis was significantly facilitated in mice with increased TrkB signaling but delayed in mutants with reduced TrkB signaling. In contrast, TrkB signaling did not influence granule cell dispersion, an important feature of this mouse model which is also observed in most MTLE patients. These results suggest that an increase in TrkB signaling, mediated by a long-lasting BDNF overexpression in the hippocampus, promotes epileptogenesis in MTLE.

Early life stress predisposes to the development of psychiatric disorders. In this context the hippocampal formation is of particular interest, because it is affected by stress on the structural and cognitive level. Since little is known how early life stress is translated on the molecular level, we mimicked early life stress in mouse models and analyzed the expression of the glycoprotein Reelin, a master molecule for development and differentiation of the hippocampus. From postnatal day 1 (P1) to P14, mouse pups were subjected to one of the following treatments: nonhandling (NH), handling (H), maternal separation (MS), and early deprivation (ED) followed by immediate (P15) or delayed (P70) real time RT-PCR analysis of reelin mRNA expression. We show that at P15, reelin mRNA levels were significantly increased in male H and ED groups when compared with the NH group. In contrast, no stress-induced alterations of reelin mRNA expression were found in female animals. This sex difference in stress-mediated stimulation of reelin expression was maintained into adulthood, since at P70 intergroup differences were still found in male, but not in female mice. On the cellular level, however, we did not find any significant differences in cell densities of Reelin-immunolabeled neurons between treatment groups or sexes, but an overall reduction of Reelin-expressing neurons in the adult hippocampus when compared to P15. To address the question whether corticosterone mediates the stress-induced up-regulation of reelin gene expression, we used age-matched hippocampal slice cultures derived from male and female mouse pups. Quantitative determination of mRNA levels revealed that corticosterone treatment significantly up-regulated reelin mRNA expression in male, but not in female hippocampi. Taken together, these results show a sex-specific regulation of reelin gene expression by early life experience, most likely mediated by corticosterone.