Intraventricular haemorrhage in premature infants: the role of immature neuronal salt and water transport.

Intraventricular hemorrhage (IVH) is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability, and/or hydrocephalus. Animal models suggest that brain tissue shrinkage with subsequent vascular stretch and tear is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for IVH are biomarkers of hypoxic-ischemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction under these conditions. This is because immature neurons express the chloride salt and water transporter NKCC1, which subserves regulatory volume increases in nonneural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic ischemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters will be diminished. As a consequence, while mature neurons swell (cytotoxic edema) immature neurons might shrink. After hypoxic-ischemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage, and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, as well as prevention of neuronal shrinkage and the ensuing IVH, in premature infants.

A Dynamic Balance between Neuronal Death and Clearance in an in Vitro Model of Acute Brain Injury.

In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.SIGNIFICANCE STATEMENT In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus, longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically expressed proteins, provide more accurate estimates of cell death than do single-time point anatomic or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

Cellular resolution contributions to ictal population signals.

OBJECTIVE: The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. METHODS: To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. RESULTS: The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. SIGNIFICANCE: This work provides a critical link between single neuron activity and population measures of seizure activity.

Brief apnea with hypoventilation reduces seizure duration and shifts seizure location for several hours in a model of severe traumatic brain injury.

OBJECTIVE: Seizures can be difficult to control in infants and toddlers. Seizures with periods of apnea and hypoventilation are common following severe traumatic brain injury (TBI). We previously observed that brief apnea with hypoventilation (A&H) in our severe TBI model acutely interrupted seizures. The current study is designed to determine the effect of A&H on subsequent seizures and whether A&H has potential therapeutic implications. METHODS: Piglets (1 week or 1 month old) received multifactorial injuries: cortical impact, mass effect, subdural hematoma, subarachnoid hemorrhage, and seizures induced with kainic acid. A&H (1 min apnea, 10 min hypoventilation) was induced either before or after seizure induction, or control piglets received subdural/subarachnoid hematoma and seizure without A&H. In an intensive care unit, piglets were sedated, intubated, and mechanically ventilated, and epidural electroencephalogram was recorded for an average of 18 h after seizure induction. RESULTS: In our severe TBI model, A&H after seizure reduced ipsilateral seizure burden by 80% compared to the same injuries without A&H. In the A&H before seizure induction group, more piglets had exclusively contralateral seizures, although most piglets in all groups had seizures that shifted location throughout the several hours of seizure. After 8-10 h, seizures transitioned to interictal epileptiform discharges regardless of A&H or timing of A&H. SIGNIFICANCE: Even brief A&H may alter traumatic seizures. In our preclinical model, we will address the possibility of hypercapnia with normoxia, with controlled intracranial pressure, as a therapeutic option for children with status epilepticus after hemorrhagic TBI.

Can in vitro studies aid in the development and use of antiseizure therapies? A report of the ILAE/AES Joint Translational Task Force.

In vitro preparations (defined here as cultured cells, brain slices, and isolated whole brains) offer a variety of approaches to modeling various aspects of seizures and epilepsy. Such models are particularly amenable to the application of anti-seizure compounds, and consequently are a valuable tool to screen the mechanisms of epileptiform activity, mode of action of known anti-seizure medications (ASMs), and the potential efficacy of putative new anti-seizure compounds. Despite these applications, all disease models are a simplification of reality and are therefore subject to limitations. In this review, we summarize the main types of in vitro models that can be used in epilepsy research, describing key methodologies as well as notable advantages and disadvantages of each. We argue that a well-designed battery of in vitro models can form an effective and potentially high-throughput screening platform to predict the clinical usefulness of ASMs, and that in vitro models are particularly useful for interrogating mechanisms of ASMs. To conclude, we offer several key recommendations that maximize the potential value of in vitro models in ASM screening. This includes the use of multiple in vitro tests that can complement each other, carefully combined with in vivo studies, the use of tissues from chronically epileptic (rather than naïve wild-type) animals, and the integration of human cell/tissue-derived preparations.

In vitro ictogenesis is stochastic at the single neuron level.

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas cannot be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed three patterns of seizure onset: (i) low amplitude fast activity; (ii) sentinel spike; and (iii) spike burst and low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal 'bad actors' (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among large populations of neurons.

Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains.

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the GABAA receptor reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl-]i) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABAA response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABAA reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.

A Pilot Randomized, Controlled, Double-Blind Trial of Bumetanide to Treat Neonatal Seizures.

OBJECTIVE: In the absence of controlled trials, treatment of neonatal seizures has changed minimally despite poor drug efficacy. We tested bumetanide added to phenobarbital to treat neonatal seizures in the first trial to include a standard-therapy control group. METHODS: A randomized, double-blind, dose-escalation design was employed. Neonates with postmenstrual age 33 to 44 weeks at risk of or with seizures were eligible. Subjects with electroencephalography (EEG)-confirmed seizures after ≥20 and <40mg/kg phenobarbital were randomized to receive additional phenobarbital with either placebo (control) or 0.1, 0.2, or 0.3mg/kg bumetanide (treatment). Continuous EEG monitoring data from ≥2 hours before to ≥48 hours after study drug administration (SDA) were analyzed for seizures. RESULTS: Subjects were randomized to treatment (n = 27) and control (n = 16) groups. Pharmacokinetics were highly variable among subjects and altered by hypothermia. The only statistically significant adverse event was diuresis in treated subjects (48% vs 13%, p = 0.02). One treated (4%) and 3 control subjects died (19%, p = 0.14). Among survivors, 2 of 26 treated subjects (8%) and 0 of 13 control subjects had hearing impairment, as did 1 nonrandomized subject. Total seizure burden varied widely, with much higher seizure burden in treatment versus control groups (median = 3.1 vs 1.2 min/h, p = 0.006). There was significantly greater reduction in seizure burden 0 to 4 hours and 2 to 4 hours post-SDA (both p < 0.01) compared with 2-hour baseline in treatment versus control groups with adjustment for seizure burden. INTERPRETATION: Although definitive proof of efficacy awaits an appropriately powered phase 3 trial, this randomized, controlled, multicenter trial demonstrated an additional reduction in seizure burden attributable to bumetanide over phenobarbital without increased serious adverse effects. Future trials of bumetanide and other drugs should include a control group and balance seizure severity. ANN NEUROL 2021;89:327-340.

Clarifications regarding bumetanide for neonatal seizures.

A recent Phase II randomized, controlled trial of bumetanide as an adjunctive treatment for neonatal seizures showed a robust efficacy signal and no evidence of toxicity. Concerns regarding bumetanide as an adjunctive anticonvulsant are addressed here. An adequately powered multi-institutional trial is needed to accurately determine efficacy.

A perfect storm: The distribution of tissue damage depends on seizure duration, hemorrhage, and developmental stage in a gyrencephalic, multi-factorial, severe traumatic brain injury model.

The pathophysiology of extensive cortical tissue destruction observed in hemispheric hypodensity, a severe type of brain injury observed in young children, is unknown. Here, we utilize our unique, large animal model of hemispheric hypodensity with multifactorial injuries and insults to understand the pathophysiology of this severe type of traumatic brain injury, testing the effect of different stages of development. Piglets developmentally similar to human infants (1 week old, "infants") and toddlers (1 month old, "toddlers") underwent injuries and insults scaled to brain volume: cortical impact, creation of mass effect, placement of a subdural hematoma, seizure induction, apnea, and hypoventilation or a sham injury while anesthetized with a seizure-permissive regimen. Piglets receiving model injuries required overnight intensive care. Hemispheres were evaluated for damage via histopathology. The pattern of damage was related to seizure duration and hemorrhage pattern in "toddlers" resulting in a unilateral hemispheric pattern of damage ipsilateral to the injuries with sparing of the deep brain regions and the contralateral hemisphere. While "infants" had the equivalent duration of seizures as "toddlers", damage was less than "toddlers", not correlated to seizure duration, and was bilateral and patchy as is often observed in human infants. Subdural hemorrhagewas associate with adjacent focal subarachnoid hemorrhage. The percentage of the hemisphere covered with subarachnoid hemorrhage was positively correlated with damage in both developmental stages. In "infants", hemorrhage over the cortex was associated with damage to the cortex with sparing of the deep gray matter regions; without hemorrhage, damage was directed to the hippocampus and the cortex was spared. "Infants" had lower neurologic scores than "toddlers". This multifactorial model of severe brain injury caused unilateral, wide-spread destruction of the cortex in piglets developmentally similar to toddlers where both seizure duration and hemorrhage covering the brain were positively correlated to tissue destruction. Inherent developmental differences may affect how the brain responds to seizure, and thus, affects the extent and pattern of damage. Study into specifically how the "infant" brain is resistant to the effects of seizure is currently underway and may identify potential therapeutic targets that may reduce evolution of tissue damage after severe traumatic brain injury.

A Proposed Mechanism for Spontaneous Transitions between Interictal and Ictal Activity.

Epileptic networks are characterized by two outputs: brief interictal spikes and rarer, more prolonged seizures. Although either output state is readily modeled in silico and induced experimentally, the transition mechanisms are unknown, in part because no models exhibit both output states spontaneously. In silico small-world neural networks were built using single-compartment neurons whose physiological parameters were derived from dual whole-cell recordings of pyramidal cells in organotypic hippocampal slice cultures that were generating spontaneous seizure-like activity. In silico, neurons were connected by abundant local synapses and rare long-distance synapses. Activity-dependent synaptic depression and gradual recovery delimited synchronous activity. Full synaptic recovery engendered interictal population spikes that spread via long-distance synapses. When synaptic recovery was incomplete, postsynaptic neurons required coincident activation of multiple presynaptic terminals to reach firing threshold. Only local connections were sufficiently dense to spread activity under these conditions. This coalesced network activity into traveling waves whose velocity varied with synaptic recovery. Seizures were comprised of sustained traveling waves that were similar to those recorded during experimental and human neocortical seizures. Sustained traveling waves occurred only when wave velocity, network dimensions, and the rate of synaptic recovery enabled wave reentry into previously depressed areas at precisely ictogenic levels of synaptic recovery. Wide-field, cellular-resolution GCamP7b calcium imaging demonstrated similar initial patterns of activation in the hippocampus, although the anatomical distribution of traveling waves of synaptic activation was altered by the pattern of synaptic connectivity in the organotypic hippocampal cultures.SIGNIFICANCE STATEMENT When computerized distributed neural network models are required to generate both features of epileptic networks (i.e., spontaneous interictal population spikes and seizures), the network structure is substantially constrained. These constraints provide important new hypotheses regarding the nature of epileptic networks and mechanisms of seizure onset.

Scalp recorded spike ripples predict seizure risk in childhood epilepsy better than spikes.

In the past decade, brief bursts of fast oscillations in the ripple range have been identified in the scalp EEG as a promising non-invasive biomarker for epilepsy. However, investigation and clinical application of this biomarker have been limited because standard approaches to identify these brief, low amplitude events are difficult, time consuming, and subjective. Recent studies have demonstrated that ripples co-occurring with epileptiform discharges ('spike ripple events') are easier to detect than ripples alone and have greater pathological significance. Here, we used objective techniques to quantify spike ripples and test whether this biomarker predicts seizure risk in childhood epilepsy. We evaluated spike ripples in scalp EEG recordings from a prospective cohort of children with a self-limited epilepsy syndrome, benign epilepsy with centrotemporal spikes, and healthy control children. We compared the rate of spike ripples between children with epilepsy and healthy controls, and between children with epilepsy during periods of active disease (active, within 1 year of seizure) and after a period of sustained seizure-freedom (seizure-free, >1 year without seizure), using semi-automated and automated detection techniques. Spike ripple rate was higher in subjects with active epilepsy compared to healthy controls (P = 0.0018) or subjects with epilepsy who were seizure-free ON or OFF medication (P = 0.0018). Among epilepsy subjects with spike ripples, each month seizure-free decreased the odds of a spike ripple by a factor of 0.66 [95% confidence interval (0.47, 0.91), P = 0.021]. Comparing the diagnostic accuracy of the presence of at least one spike ripple versus a classic spike event to identify group, we found comparable sensitivity and negative predictive value, but greater specificity and positive predictive value of spike ripples compared to spikes (P = 0.016 and P = 0.006, respectively). We found qualitatively consistent results using a fully automated spike ripple detector, including comparison with an automated spike detector. We conclude that scalp spike ripple events identify disease and track with seizure risk in this epilepsy population, using both semi-automated and fully automated detection methods, and that this biomarker outperforms analysis of spikes alone in categorizing seizure risk. These data provide evidence that spike ripples are a specific non-invasive biomarker for seizure risk in benign epilepsy with centrotemporal spikes and support future work to evaluate the utility of this biomarker to guide medication trials and tapers in these children and predict seizure risk in other at-risk populations.

Epileptiform activity in traumatic brain injury predicts post-traumatic epilepsy.

We hypothesize that epileptiform abnormalities (EAs) in the electroencephalogram (EEG) during the acute period following traumatic brain injury (TBI) independently predict first-year post-traumatic epilepsy (PTE1 ). We analyze PTE1 risk factors in two cohorts matched for TBI severity and age (n = 50). EAs independently predict risk for PTE1 (odds ratio [OR], 3.16 [0.99, 11.68]); subdural hematoma is another independent risk factor (OR, 4.13 [1.18, 39.33]). Differences in EA rates are apparent within 5 days following TBI. Our results suggest that increased EA prevalence identifies patients at increased risk for PTE1 , and that EAs acutely post-TBI can identify patients most likely to benefit from antiepileptogenesis drug trials. Ann Neurol 2018;83:858-862.

Standards for data acquisition and software-based analysis of in vivo electroencephalography recordings from animals. A TASK1-WG5 report of the AES/ILAE Translational Task Force of the ILAE.

Electroencephalography (EEG)-the direct recording of the electrical activity of populations of neurons-is a tremendously important tool for diagnosing, treating, and researching epilepsy. Although standard procedures for recording and analyzing human EEG exist and are broadly accepted, there are no such standards for research in animal models of seizures and epilepsy-recording montages, acquisition systems, and processing algorithms may differ substantially among investigators and laboratories. The lack of standard procedures for acquiring and analyzing EEG from animal models of epilepsy hinders the interpretation of experimental results and reduces the ability of the scientific community to efficiently translate new experimental findings into clinical practice. Accordingly, the intention of this report is twofold: (1) to review current techniques for the collection and software-based analysis of neural field recordings in animal models of epilepsy, and (2) to offer pertinent standards and reporting guidelines for this research. Specifically, we review current techniques for signal acquisition, signal conditioning, signal processing, data storage, and data sharing, and include applicable recommendations to standardize collection and reporting. We close with a discussion of challenges and future opportunities, and include a supplemental report of currently available acquisition systems and analysis tools. This work represents a collaboration on behalf of the American Epilepsy Society/International League Against Epilepsy (AES/ILAE) Translational Task Force (TASK1-Workgroup 5), and is part of a larger effort to harmonize video-EEG interpretation and analysis methods across studies using in vivo and in vitro seizure and epilepsy models.

Evolution of Network Synchronization during Early Epileptogenesis Parallels Synaptic Circuit Alterations.

In secondary epilepsy, a seizure-prone neural network evolves during the latent period between brain injury and the onset of spontaneous seizures. The nature of the evolution is largely unknown, and even its completeness at the onset of seizures has recently been challenged by measures of gradually decreasing intervals between subsequent seizures. Sequential calcium imaging of neuronal activity, in the pyramidal cell layer of mouse hippocampal in vitro preparations, during early post-traumatic epileptogenesis demonstrated rapid increases in the fraction of neurons that participate in interictal activity. This was followed by more gradual increases in the rate at which individual neurons join each developing seizure, the pairwise correlation of neuronal activities as a function of the distance separating the pair, and network-wide measures of functional connectivity. These data support the continued evolution of synaptic connectivity in epileptic networks beyond the latent period: early seizures occur when recurrent excitatory pathways are largely polysynaptic, while ongoing synaptic remodeling after the onset of epilepsy enhances intranetwork connectivity as well as the onset and spread of seizure activity.

Failure to detect human papillomavirus in focal cortical dysplasia type IIb.

OBJECTIVE: Recent studies have reported evidence of human papillomavirus 16 (HPV-16) in a very high proportion of pathological specimens of focal cortical dysplasia type IIb, but not in control specimens, motivating the proposal that viral infection during fetal development may play a causal role in the pathogenesis of focal cortical dysplasias. However, the significance of the association between HPV infection and focal cortical dysplasia type IIb, and its reproducibility across surgical centers, remain unclear. Here we sought evidence for HPV-16 in an independent cohort of surgical specimens. METHODS: We identified 14 specimens of focal cortical dysplasia type IIb from a single surgical center between 1995 and 2013. Multiple methods were used to establish presence or absence of HPV, including DNA polymerase chain reaction, conventional in situ hybridization, chromogenic in situ hybridization, and immunohistochemistry for p16. RESULTS: We found no conclusive evidence of HPV in any of the specimens. All but 1 of the cases were negative by >1 method. INTERPRETATION: These results raise questions about the prevalence of HPV infection in focal cortical dysplasias and about its potential importance as a causative agent.

PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.

mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an anti-epileptogenic, or merely anticonvulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long-term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with lactate dehydrogenase (LDH) release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are as follows: (1) the organotypic hippocampal culture model of post-traumatic epilepsy comprises a rapid assay of anti-epileptogenic and neuroprotective activities and, in this model (2) mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.

Novel determinants of the neuronal Cl(-) concentration.

It is now a well-accepted view that cation-driven Cl(-) transporters in neurons are involved in determining the intracellular Cl(-) concentration. In the present review, we propose that additional factors, which are often overlooked, contribute substantially to the Cl(-) gradient across neuronal membranes. After briefly discussing the data supporting and opposing the role of cation-chloride cotransporters in regulating Cl(-), we examine the participation of the following factors in the formation of the transmembrane Cl(-) gradient: (i) fixed 'Donnan' charges inside and outside the cell; (ii) the properties of water (free vs. bound); and (iii) water transport through the cotransporters. We demonstrate a steep relationship between intracellular Cl(-) and the concentration of fixed negative charges on macromolecules. We show that in the absence of water transport through the K(+)-Cl(-) cotransporter, a large osmotic gradient builds at concentrations below or above a set value of 'Donnan' charges, and show that at any value of these fixed charges, the reversal potential for Cl(-) equates that of K(+). When the movement of water across the membrane is a source of free energy, it is sufficient to modify the movement of Cl(-) through the cotransporter. In this scenario, the reversal potential for Cl(-) does not closely follow that of K(+). Furthermore, our simulations demonstrate that small differences in the availability of freely diffusible water between inside and outside the cell greatly affect the Cl(-) reversal potential, particularly when osmolar transmembrane gradients are minimized, for example by idiogenic osmoles. We also establish that the presence of extracellular charges has little effect on the chloride reversal potential, but greatly affects the effective inhibitory conductance for Cl(-). In conclusion, our theoretical analysis of the presence of fixed anionic charges and water bound on macromolecules inside and outside the cell greatly impacts both Cl(-) gradient and Cl(-) conductance across neuronal membranes.

Soluble pathological tau in the entorhinal cortex leads to presynaptic deficits in an early Alzheimer's disease model.

Neurofibrillary tangles (NFTs), a hallmark of Alzheimer's disease, are intracellular silver and thioflavin S-staining aggregates that emerge from earlier accumulation of phospho-tau in the soma. Whether soluble misfolded but nonfibrillar tau disrupts neuronal function is unclear. Here we investigate if soluble pathological tau, specifically directed to the entorhinal cortex (EC), can cause behavioral or synaptic deficits. We studied rTgTauEC transgenic mice, in which P301L mutant human tau overexpressed primarily in the EC leads to the development of tau pathology, but only rare NFT at 16 months of age. We show that the early tau lesions are associated with nearly normal performance in contextual fear conditioning, a hippocampal-related behavior task, but more robust changes in neuronal system activation as marked by Arc induction and clear electrophysiological defects in perforant pathway synaptic plasticity. Electrophysiological changes were likely due to a presynaptic deficit and changes in probability of neurotransmitter release. The data presented here support the hypothesis that misfolded and hyperphosphorylated tau can impair neuronal function within the entorhinal-hippocampal network, even prior to frank NFT formation and overt neurodegeneration.

What is the clinical relevance of in vitro epileptiform activity?

In vitro preparations provide an exceptionally rapid, flexible, and accessible approach to long-standing problems in epilepsy research including ictogenesis, epileptogenesis, and drug resistance. Acute slices suffer from a reduction in network connectivity that has traditionally been compensated through the application of acute convulsants. The utility and limitations of this approach have become clear over time and are discussed here. Other approaches such as organotypic slice preparations demonstrate the full spectrum of spontaneous epileptic activity and more closely mimic human responses to anticonvulsants, including the development of drug resistance. Newly developed transgenic and vector expression systems for fluorophores, optogenetics, and orphan receptors are being coupled with advances in imaging and image analysis. These developments have created the capacity to rapidly explore many new avenues of epilepsy research such as vascular, astrocytic and mitochondrial contributions to epileptogenesis. Rigorous study design as well as close collaboration with in vivo laboratories and clinical investigators will accelerate the translation of the exciting discoveries that will be revealed by these new techniques.