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Mesial temporal lobe epilepsy (MTLE), the most common form of focal epilepsy in adults, is often refractory to medication and associated with hippocampal sclerosis. Deep brain stimulation represents an alternative treatment option for drug-resistant patients who are ineligible for resective brain surgery. In clinical practice, closed-loop stimulation at high frequencies is applied to interrupt ongoing seizures, yet with a high incidence of false detections, the drawback of delayed seizure-suppressive intervention and limited success in sclerotic tissue. More recently, hippocampal low-frequency stimulation (LFS) has been shown to reduce excitability in clinical settings and prevent seizures in experimental MTLE when applied continuously. However, as the hippocampus is important for navigation and memory, it would be beneficial to stimulate it only on-demand to reduce its exposure to LFS pulses, and to investigate LFS-related effects on cognition.Using the intrahippocampal kainate mouse model, which recapitulates the key features of MTLE, we developed an on-demand LFS setup and investigated its effects on spontaneous seizure activity and hippocampal function. Specifically, our online detection algorithm monitored epileptiform activity in hippocampal local field potential recordings and identified short epileptiform bursts preceding focal seizure clusters, triggering hippocampal LFS to stabilize the network state. In addition, we investigated the acute influence of LFS on behavioral performance, including anxiety-like behavior in the light-dark box test, spatial and non-spatial memory in the object location memory and novel object recognition test, as well as spatial navigation and long-term memory in the Barnes maze.Compared to open-loop stimulation protocols, on-demand LFS was more efficient in preventing focal seizure clusters, as the strong anti-epileptic effect was achieved with a reduced stimulation load. In behavioral tests, chronically epileptic mice were as mobile as healthy controls but showed increased anxiety, an altered spatial learning strategy and impaired memory performance. Most importantly, our experiments ruled out deleterious effects of hippocampal LFS on cognition and even showed alleviation of deficits in long-term memory recall. Taken together, our findings may provide a promising alternative to current therapies, overcoming some of their major limitations, and inspire further investigation of LFS for seizure control in MTLE.

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.

The reticulated flatwoods salamander (Ambystoma bishopi), an endangered species endemic to the longleaf-pine savanna ecosystem of the southeastern U.S., persists in a small number of remnant habitat patches. Breeding ponds and associated populations are threatened by habitat loss, degradation, and fragmentation stemming from fire suppression and land conversion. Understanding influences on population dynamics and genetic diversity will help inform recovery efforts for this and other pond-breeding amphibians. We used 9 microsatellite loci to characterize population structure, migration, and genetic diversity of juvenile (larvae and metamorphs) A. bishopi (n = 607) sampled from thirteen breeding ponds across two breeding seasons. Temporal genetic variation between annual cohorts was minimal compared to spatial variation among locations. The primary genetic subdivision was between two regional groups of breeding ponds (i.e. metapopulations), which were located more than 10 km from each other. Yet even within metapopulations, genetic isolation-by-distance was pronounced, and estimated migration rates among ponds separated by > 400 m were very low. Genetic diversity was positively correlated with the area of suitable breeding habitat in a pond and negatively correlated with distance to other occupied ponds. The effective number of breeders typically was

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.

Selective loss of inhibitory interneurons (INs) that promotes a shift toward an excitatory predominance may have a critical impact on the generation of epileptic activity. While research on mesial temporal lobe epilepsy (MTLE) has mostly focused on hippocampal changes, including IN loss, the subiculum as the major output region of the hippocampal formation has received comparatively little attention. Although it has been shown to occupy a key position in the epileptic network, data on cellular changes in the subiculum are controversial. Using the intrahippocampal kainate (KA) mouse model for MTLE, which recapitulates main features of human MTLE such as hippocampal sclerosis and granule cell dispersion following status epilepticus (SE), we identified cell loss in the subiculum and quantified changes in specific IN subpopulations along its dorsoventral axis.We performed intrahippocampal recordings, FluoroJade C-staining for degenerating neurons shortly after SE and immunohistochemistry for neuronal nuclei (NeuN), parvalbumin (PV), neuropeptide Y (NPY), calretinin (CR) and calbindin (CB), and in situ hybridization for glutamic acid decarboxylase (Gad) 67 mRNA at 21 days after KA.We observed remarkable cell loss in the ipsilateral subiculum shortly after SE which was reflected in lowered density of NeuN+ cells in the chronic stage when epileptic activity could be measured in the subiculum concomitant with the hippocampus. We show a position-dependent reduction of Gad67-expressing INs by ~50% which particularly affected the PV- and to a lesser extent CR-expressing INs, whereas CB-expressing cells were unchanged.Interestingly, the density of NPY-positive neurons was increased, but double-labeling for Gad67 mRNA expression revealed that rather a de novo expression of NPY in non-GABAergic cells instead of IN changes underlay this increase.Our data reveal a position- and cell type-specific vulnerability of subicular INs in MTLE which might contribute to hyperexcitability of the subiculum, as reflected in the occurrence of epileptic activity.Keypoints (3-5)The subiculum develops epileptic activity after intrahippocampal kainate injection in miceGad67-mRNA expressing interneurons are reduced in the subiculum in the intrahippocampal kainate model for mesial temporal lobe epilepsyParvalbumin- and calretinin-expressing interneurons are particularly vulnerable in epilepsyNeuropeptide Y upregulation in non-GABAergic cells in the subiculum indicates compensatory processes

Reticulated flatwoods salamander (Ambystoma bishopi) populations began decreasing dramatically in the 1900s. Contemporary populations are small, isolated, and may be susceptible to inbreeding and reduced adaptive potential because of low genetic variation. Genetic variation at immune genes is especially important as it influences disease susceptibility and adaptation to emerging infectious pathogens, a central conservation concern for declining amphibians. We collected samples from across the extant range of this salamander to examine genetic variation at major histocompatibility complex (MHC) class Iα and IIβ exons as well as the mitochondrial control region. We screened tail or toe tissue for ranavirus, a pathogen associated with amphibian declines worldwide. Overall, we found low MHC variation when compared to other amphibian species and did not detect ranavirus at any site. MHC class Iα sequencing revealed only three alleles with a nucleotide diversity of 0.001, while MHC class IIβ had five alleles with a with nucleotide diversity of 0.004. However, unique variation still exists across this species' range with private alleles at three sites. Unlike MHC diversity, mitochondrial variation was comparable to levels estimated for other amphibians with nine haplotypes observed, including one haplotype shared across all sites. We hypothesize that a combination of a historic disease outbreak and a population bottleneck may have contributed to low MHC diversity while maintaining higher levels of mitochondrial DNA variation. Ultimately, MHC data indicated that the reticulated flatwoods salamander may be at an elevated risk from infectious diseases due to low levels of immunogenetic variation necessary to combat novel pathogens.

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.

In conservation science, metapopulation models are frequently used to explore the spatial dynamics of organisms, and ultimately guide management decisions for threatened and endangered species. Pond‐breeding amphibians are often touted as a classic example of metapopulation structure. However, empirical assessment of this assumption is lacking for many species owing to the prohibitively high costs of movement studies. Our aim was to use spatially explicit occupancy models to evaluate metapopulation dynamics in an endangered amphibian. First, dynamic, spatially explicit occupancy‐based metapopulation models were fitted to 8 years of presence–absence data for the endangered reticulated flatwoods salamander, Ambystoma bishopi. Models were constructed to evaluate the spatial distribution of salamanders across the landscape, rate of patch turnover, and connectivity between patches. Second, the same data were analysed adopting an eigenvector mapping approach and spatial correlograms to investigate habitat characteristics associated with occupancy state. The second approach also yields an independent measure of patch connectivity to corroborate findings from the dynamic model. Both models predicted a steep drop‐off in connectivity with increasing isolation, and no evidence was found of interactions between wetlands separated by distances greater than 1.5 km. The best predictor of salamander occupancy status was the amount of understorey vegetation in wetland basins. Taken together, the two modelling approaches provide complementary information regarding the key determinants of occupancy in the focal species. Furthermore, several results directly translate to management recommendations. Connectivity between distant sites is low, and thus management units should be demarcated accordingly. Understorey vegetation represents egg‐laying habitat and refugia for larvae, and hence should be targeted in future restoration efforts. The methodologies employed in this paper have broad applicability to studies of pond‐breeding amphibians, particularly in situations where direct measures of dispersal are unobtainable.

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.

Electrical burst stimulation rescues movement effectively in parkinsonian mice

Gopherus polyphemus (Gopher Tortoise) populations historically occurred throughout much of the southeastern Coastal Plain, and burrows created by this species provide refugia for a large suite of commensal species. Our objective was to evaluate the physical degradation of Gopher Tortoise burrows over time. We provide burrow-status information from initial and follow-up surveys of a low-density population of Gopher Tortoises across Eglin Air Force Base (Eglin), FL, a large landscape with very deep, sandy soils. Approximately 63% (n = 79) of active and/or inactive burrows were collapsed, filled in, or substantially degraded after 2 y, 82% (n = 65) after 3 y, and 100% (n = 19) after 5 y. Compared to results from a similar study with different soils, burrows degraded more quickly on Eglin. Our results on burrow longevity can inform interpretation of Gopher Tortoise surveys and aid in predicting temporal availability of burrows for commensals.

In the new 2017 WHO classification, a reduction of the high number of entities of salivary carcinomas was implemented. There is only one new carcinoma entity: secretory carcinoma. There is a slight increase of reactive and benign entities by the inclusion of rare and well-established, but so far not included, lesions. Furthermore, there are some conceptual changes and pure changes in terminology. The impact of molecular findings is increasing and is so far restricted to diagnostic aspects.

Ephemeral wetlands provide breeding habitat for many amphibian species, and wetland hydrology plays a crucial role in determining amphibian breeding success. We discuss the potential influence of recession rates (i.e., rate of water level decline) and empirically evaluate them in wetlands inhabited by the endangered reticulated flatwoods salamander (Ambystoma bishopi). Rapid water level declines are potentially problematic for reticulated flatwoods salamanders because this species has a long development period, with metamorphosis generally occurring from March to May when groundwater losses are combined with high evapotranspiration rates. To evaluate magnitude, variability, and drivers of recession rates, we monitored water levels in 33 wetlands in the Florida panhandle and examined recession rates during the flatwoods salamander reproductive period. After controlling for the effects of specific yield, standardized recession rates were, on average, 3.9 times daily potential evapotranspiration rates, suggesting that groundwater fluxes are an important driver of water level declines in these wetlands. Standardized recession rates were variable across the landscape and increased with decreasing wetland size, indicating that larger wetlands are often hydrologically more suitable for flatwoods salamanders. This work points to these and other controls on wetland recession rates and their role in regulating amphibian reproductive success.

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.

Ephemeral wetlands surrounded by Pinus palustris (Longleaf Pine) flatwoods support diverse herpetofaunal communities and provide important breeding habitat for many species. We sampled herpetofauna in 3 pine flatwoods wetlands on Eglin Air Force Base, Okaloosa County, FL, over 2 time periods (1 wetland [1] from 1993 to1995 and 2 wetlands [2 and 3] from 2010 to 2015) using drift fences that completely encircled each wetland. We documented 37, 46, and 43 species of amphibians and reptiles at wetlands 1, 2, and 3, respectively. Herpetofaunal communities were remarkably similar across all 3 wetlands (Sorenson Index values > 0.97) despite sampling that occurred 15–20 years apart on wetlands located approximately 10 km apart. Ambystoma bishopi (Reticulated Flatwoods Salamander), Pseudacris ornata (Ornate Chorus Frog), and Eurycea quadridigitata (Dwarf Salamander), all species of conservation concern, were captured at all 3 wetlands, indicating that these wetlands provide habitat for specialist species....

Predator–prey interactions play an important role in structuring larval anuran communities in ephemeral wetlands. The type of interaction often depends on the predator species and the complexity of the aquatic habitat. We experimentally evaluated the effects of Panhandle Crayfish (Procambarus evermanni) predation on larval Southern Leopard Frogs (Lithobates sphenocephalus) and larval Ornate Chorus Frogs (Pseudacris ornata). We performed separate experiments for each anuran species and used multiple vegetation treatments to examine whether vegetation could provide refugia from crayfish predation. Our results indicate that crayfish are effective predators of anuran larvae regardless of the amount of vegetation present. Encounters between tadpoles and crayfish often resulted in nonlethal tail injury for tadpoles, suggesting that crayfish predation is prominent in ephemeral wetland communities. Leopard frog tadpoles in predator treatments also grew larger than their counterparts in nonpredator treatm...

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.

Phase-contrast micro-tomography using synchrotron radiation has yielded superior soft tissue visualization down to the sub-cellular level. The isotropic spatial resolution down to about one micron is comparable to the one of histology. The methods, however, provide different physical quantities and are thus complementary, also allowing for the extension of histology into the third dimension. To prepare for cross-sectional animal studies on epilepsy, we have standardized the specimen’s preparation and scanning procedure for mouse brains, so that subsequent histology remains entirely unaffected and scanning of all samples (n = 28) is possible in a realistic time frame. For that, we have scanned five healthy and epileptic mouse brains at the ID19 beamline, ESRF, Grenoble, France, using grating- and propagation-based phase contrast micro-tomography. The resulting datasets clearly show the cortex, ventricular system, thalamus, hypothalamus, and hippocampus. Our focus is on the latter, having planned kainate-induced epilepsy experiments. The cell density and organization in the dentate gyrus and Ammon’s horn region were clearly visualized in control animals. This proof of principle was required to initiate experiment. The resulting three-dimensional data have been correlated to histology. The goal is a brain-wide quantification of cell death or structural reorganization associated with epilepsy as opposed to histology alone that represents small volumes of the total brain only. Thus, the proposed technique bears the potential to correlate the gold standard in analysis with independently obtained data sets. Such an achievement also fuels interest for other groups in neuroscience research to closely collaborate with experts in phase micro-tomography.

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2-specific markers Purkinje cell protein 4 (PCP4) and regulator of G-Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin-expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE-associated pathological restructuring.