Kir4.1 channels contribute to astrocyte CO2/H+-sensitivity and the drive to breathe.

Astrocytes in the retrotrapezoid nucleus (RTN) stimulate breathing in response to CO2/H+, however, it is not clear how these cells detect changes in CO2/H+. Considering Kir4.1/5.1 channels are CO2/H+-sensitive and important for several astrocyte-dependent processes, we consider Kir4.1/5.1 a leading candidate CO2/H+ sensor in RTN astrocytes. To address this, we show that RTN astrocytes express Kir4.1 and Kir5.1 transcripts. We also characterized respiratory function in astrocyte-specific inducible Kir4.1 knockout mice (Kir4.1 cKO); these mice breathe normally under room air conditions but show a blunted ventilatory response to high levels of CO2, which could be partly rescued by viral mediated re-expression of Kir4.1 in RTN astrocytes. At the cellular level, astrocytes in slices from astrocyte-specific inducible Kir4.1 knockout mice are less responsive to CO2/H+ and show a diminished capacity for paracrine modulation of respiratory neurons. These results suggest Kir4.1/5.1 channels in RTN astrocytes contribute to respiratory behavior.

Traumatic brain injury disrupts state-dependent functional cortical connectivity in a mouse model.

Traumatic brain injury (TBI) is the leading cause of death in young people and can cause cognitive and motor dysfunction and disruptions in functional connectivity between brain regions. In human TBI patients and rodent models of TBI, functional connectivity is decreased after injury. Recovery of connectivity after TBI is associated with improved cognition and memory, suggesting an important link between connectivity and functional outcome. We examined widespread alterations in functional connectivity following TBI using simultaneous widefield mesoscale GCaMP7c calcium imaging and electrocorticography (ECoG) in mice injured using the controlled cortical impact (CCI) model of TBI. Combining CCI with widefield cortical imaging provides us with unprecedented access to characterize network connectivity changes throughout the entire injured cortex over time. Our data demonstrate that CCI profoundly disrupts functional connectivity immediately after injury, followed by partial recovery over 3 weeks. Examining discrete periods of locomotion and stillness reveals that CCI alters functional connectivity and reduces theta power only during periods of behavioral stillness. Together, these findings demonstrate that TBI causes dynamic, behavioral state-dependent changes in functional connectivity and ECoG activity across the cortex.

Cell type-specific gene expression dynamics during human brain maturation.

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. To date, studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-Sseq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 54 cell subtypes, which we verify with spatial transcriptomics. We explore the differences in cell states between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during a critical period of its development and contribute to global efforts to build an inclusive cell map of the brain.

How parasitic larvae affect the brain.

The release of the neurotransmitter glutamate by the parasitic tapeworm Taenia solium appears to be implicated in the pathophysiology of a widespread, but neglected, form of adult-onset epilepsy.

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.

Cortical Parvalbumin-Positive Interneuron Development and Function Are Altered in the APC Conditional Knockout Mouse Model of Infantile and Epileptic Spasms Syndrome.

Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.

Astrocytic BDNF signaling within the ventromedial hypothalamus regulates energy homeostasis.

Brain-derived neurotrophic factor (BDNF) is essential for maintaining energy and glucose balance within the central nervous system. Because the study of its metabolic actions has been limited to effects in neuronal cells, its role in other cell types within the brain remains poorly understood. Here we show that astrocytic BDNF signaling within the ventromedial hypothalamus (VMH) modulates neuronal activity in response to changes in energy status. This occurs via the truncated TrkB.T1 receptor. Accordingly, either fasting or central BDNF depletion enhances astrocytic synaptic glutamate clearance, thereby decreasing neuronal activity in mice. Notably, selective depletion of TrkB.T1 in VMH astrocytes blunts the effects of energy status on excitatory transmission, as well as on responses to leptin, glucose and lipids. These effects are driven by increased astrocytic invasion of excitatory synapses, enhanced glutamate reuptake and decreased neuronal activity. We thus identify BDNF/TrkB.T1 signaling in VMH astrocytes as an essential mechanism that participates in energy and glucose homeostasis.

Neuronal activity drives pathway-specific depolarization of peripheral astrocyte processes.

Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte-neuron interaction.

Novel Approaches to Prevent Epileptogenesis After Traumatic Brain Injury.

Traumatic brain injury (TBI) is defined as an alteration in brain function or other evidence of brain pathology caused by an external force. When epilepsy develops following TBI, it is known as post-traumatic epilepsy (PTE). PTE occurs in a subset of patients suffering from different types and severities of TBI, occurs more commonly following severe injury, and greatly impacts the quality of life for patients recovering from TBI. Similar to other types of epilepsy, PTE is often refractory to drug treatment with standard anti-seizure drugs. No therapeutic approaches have proven successful in the clinic to prevent the development of PTE. Therefore, novel treatment strategies are needed to stop the development of PTE and improve the quality of life for patients after TBI. Interestingly, TBI represents an excellent clinical opportunity for intervention to prevent epileptogenesis as typically the time of initiation of epileptogenesis (i.e., TBI) is known, the population of at-risk patients is large, and animal models for preclinical studies of mechanisms and treatment targets are available. If properly identified and treated, there is a true opportunity to prevent epileptogenesis after TBI and stop seizures from ever happening. With that goal in mind, here we review previous attempts to prevent PTE both in animal studies and in humans, we examine how biomarkers could enable better-targeted therapeutics, and we discuss how genetic variation may predispose individuals to PTE. Finally, we highlight exciting new advances in the field that suggest that there may be novel approaches to prevent PTE that should be considered for further clinical development.

Identification of clinically relevant biomarkers of epileptogenesis - a strategic roadmap.

Onset of many forms of epilepsy occurs after an initial epileptogenic insult or as a result of an identified genetic defect. Given that the precipitating insult is known, these epilepsies are, in principle, amenable to secondary prevention. However, development of preventive treatments is difficult because only a subset of individuals will develop epilepsy and we cannot currently predict which individuals are at the highest risk. Biomarkers that enable identification of these individuals would facilitate clinical trials of potential anti-epileptogenic treatments, but no such prognostic biomarkers currently exist. Several putative molecular, imaging, electroencephalographic and behavioural biomarkers of epileptogenesis have been identified, but clinical translation has been hampered by fragmented and poorly coordinated efforts, issues with inter-model reproducibility, study design and statistical approaches, and difficulties with validation in patients. These challenges demand a strategic roadmap to facilitate the identification, characterization and clinical validation of biomarkers for epileptogenesis. In this Review, we summarize the state of the art with respect to biomarker research in epileptogenesis and propose a five-phase roadmap, adapted from those developed for cancer and Alzheimer disease, that provides a conceptual structure for biomarker research.

Cells That "Fire Together, Wire Together", but Do They Transcribe Together in Epilepsy?

[Box: see text].

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus.

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20-25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

In vivo KPT-350 treatment decreases cortical hyperexcitability following traumatic brain injury.

PRIMARY OBJECTIVE: We tested whether KPT-350, a novel selective inhibitor of nuclear export, could attenuate cortical network hyperexcitability, a major risk factor for developing post-traumatic epilepsy (PTE) following traumatic brain injury (TBI). RESEARCH DESIGN: All mice in this study underwent TBI and were subsequently treated with either KPT-350 or vehicle during the post-injury latent period. Half of the animal cohort was used for electrophysiology while the other half was used for immunohistochemical analysis. METHODS AND PROCEDURES: TBI was induced using the controlled cortical impact (CCI) model. Cortical network activity was recorded by evoking field potentials from deep layers of the cortex and analyzed using Matlab software. Immunohistochemistry coupled with fluorescence microscopy and Image J analysis detected changes in neuronal and glial markers. MAIN OUTCOMES AND RESULTS: KPT-350 attenuated TBI-associated epileptiform activity and restored disrupted input-output responses in cortical brain slices. In vivo KPT-350 treatment reduced the loss of parvalbumin-(+) GABAergic interneurons after CCI but did not significantly change CCI-induced loss of somatostatin-(+) GABAergic interneurons, nor did it reduce reactivity of GFAP and Iba1 glial markers. CONCLUSION: KPT-350 can prevent cortical hyperexcitability and reduce the loss of parvalbumin-(+) GABAergic inhibitory neurons, making it a potential therapeutic option for preventing PTE.

The adenosine A1 receptor agonist WAG 994 suppresses acute kainic acid-induced status epilepticus in vivo.

Status epilepticus (SE) is a neurological emergency characterized by continuous seizure activity lasting longer than 5 min, often with no recovery between seizures (Trinka et al., 2015). SE is refractory to benzodiazepine and second-line treatments in about 30% cases. Novel treatment approaches are urgently needed as refractory SE is associated with mortality rates of up to 70%. Robust adenosinergic anticonvulsant effects have been known for decades, but translation into seizure treatments was hampered by cardiovascular side effects. However, the selective adenosine A1 receptor agonist SDZ WAG 994 (WAG) displays diminished cardiovascular side effects compared to classic A1R agonists and was safely administered systemically in human clinical trials. Here, we investigate the anticonvulsant efficacy of WAG in vitro and in vivo. WAG robustly inhibited high-K+-induced continuous epileptiform activity in rat hippocampal slices (IC50 = 52.5 nM). Importantly, WAG acutely suppressed SE in vivo induced by kainic acid (20 mg/kg i.p.) in mice. After SE was established, mice received three i.p. injections of WAG or diazepam (DIA, 5 mg/kg). Interestingly, DIA did not attenuate SE while the majority of WAG-treated mice (1 mg/kg) were seizure-free after three injections. Anticonvulsant effects were retained when a lower dose of WAG (0.3 mg/kg) was used. Importantly, all WAG-treated mice survived kainic acid induced SE. In summary, we report for the first time that an A1R agonist with an acceptable human side-effect profile can acutely suppress established SE in vivo. Our results suggest that WAG stops or vastly attenuates SE while DIA fails to mitigate SE in this model.

Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake.

Genetically encoded fluorescent glutamate indicators (iGluSnFRs) enable neurotransmitter release and diffusion to be visualized in intact tissue. Synaptic iGluSnFR signal time courses vary widely depending on experimental conditions, often lasting 10-100 times longer than the extracellular lifetime of synaptically released glutamate estimated with uptake measurements. iGluSnFR signals typically also decay much more slowly than the unbinding kinetics of the indicator. To resolve these discrepancies, here we have modeled synaptic glutamate diffusion, uptake and iGluSnFR activation to identify factors influencing iGluSnFR signal waveforms. Simulations suggested that iGluSnFR competes with transporters to bind synaptically released glutamate, delaying glutamate uptake. Accordingly, synaptic transporter currents recorded from iGluSnFR-expressing astrocytes in mouse cortex were slower than those in control astrocytes. Simulations also suggested that iGluSnFR reduces free glutamate levels in extrasynaptic spaces, likely limiting extrasynaptic receptor activation. iGluSnFR and lower affinity variants, nonetheless, provide linear indications of vesicle release, underscoring their value for optical quantal analysis.

A Tale of two Networks-Glial Contributions to Generalized Seizures.

[Box: see text].

Epilepsy Benchmarks Area I: Understanding the Causes of the Epilepsies and Epilepsy-Related Neurologic, Psychiatric, and Somatic Conditions.

The 2014 NINDS Benchmarks for Epilepsy Research included area I: Understand the causes of the epilepsies and epilepsy-related neurologic, psychiatric, and somatic conditions. In preparation for the 2020 Curing Epilepsies Conference, where the Benchmarks will be revised, this review will cover scientific progress toward that Benchmark, with emphasize on studies since 2016.