Synthesis and Characterization of a Novel Concentration-Independent Fluorescent Chloride Indicator, ABP-Dextran, Optimized for Extracellular Chloride Measurement.

GABA, the primary inhibitory neurotransmitter, stimulates GABAA receptors (GABAARs) to increase the chloride conductance of the cytosolic membrane. The driving forces for membrane chloride currents are determined by the local differences between intracellular and extracellular chloride concentrations (Cli and Clo, respectively). While several strategies exist for the measurement of Cli, the field lacks tools for non-invasive measurement of Clo. We present the design and development of a fluorescent lifetime imaging (FLIM)-compatible small molecule, N(4-aminobutyl)phenanthridiunium (ABP) with the brightness, spectral features, sensitivity to chloride, and selectivity versus other anions to serve as a useful probe of Clo. ABP can be conjugated to dextran to ensure extracellular compartmentalization, and a second chloride-insensitive counter-label can be added for ratiometric imaging. We validate the utility of this novel sensor series in two sensor concentration-independent modes: FLIM or ratiometric intensity-based imaging.

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.

Effects of AFQ056 on language learning in fragile X syndrome.

BACKGROUNDFXLEARN, the first-ever large multisite trial of effects of disease-targeted pharmacotherapy on learning, was designed to explore a paradigm for measuring effects of mechanism-targeted treatment in fragile X syndrome (FXS). In FXLEARN, the effects of metabotropic glutamate receptor type 5 (mGluR5) negative allosteric modulator (NAM) AFQ056 on language learning were evaluated in 3- to 6-year-old children with FXS, expected to have more learning plasticity than adults, for whom prior trials of mGluR5 NAMs have failed.METHODSAfter a 4-month single-blind placebo lead-in, participants were randomized 1:1 to AFQ056 or placebo, with 2 months of dose optimization to the maximum tolerated dose, then 6 months of treatment during which a language-learning intervention was implemented for both groups. The primary outcome was a centrally scored videotaped communication measure, the Weighted Communication Scale (WCS). Secondary outcomes were objective performance-based and parent-reported cognitive and language measures.RESULTSFXLEARN enrolled 110 participants, randomized 99, and had 91 who completed the placebo-controlled period. Although both groups made language progress and there were no safety issues, the change in WCS score during the placebo-controlled period was not significantly different between the AFQ056 and placebo-treated groups, nor were there any significant between-group differences in change in any secondary measures.CONCLUSIONDespite the large body of evidence supporting use of mGluR5 NAMs in animal models of FXS, this study suggests that this mechanism of action does not translate into benefit for the human FXS population and that better strategies are needed to determine which mechanisms will translate from preclinical models to humans in genetic neurodevelopmental disorders.TRIAL REGISTRATIONClincalTrials.gov NCT02920892.FUNDING SOURCESNeuroNEXT network NIH grants U01NS096767, U24NS107200, U24NS107209, U01NS077323, U24NS107183, U24NS107168, U24NS107128, U24NS107199, U24NS107198, U24NS107166, U10NS077368, U01NS077366, U24NS107205, U01NS077179, and U01NS077352; NIH grant P50HD103526; and Novartis IIT grant AFQ056X2201T for provision of AFQ056.

Effect of neonatal seizure burden and etiology on the long-term outcome: data from a randomized, controlled trial.

Background: Neonatal seizures are common, but the impact of neonatal seizures on long-term neurologic outcome remains unclear. We addressed this question by analyzing data from an early-phase controlled trial of bumetanide to treat neonatal seizures. Methods: Neonatal seizure burden was calculated from continuous video-EEG data. Neurologic outcome was determined by standardized developmental tests and post-neonatal seizure recurrence. Results: Of 111 enrolled neonates, 43 were randomized to treatment or control groups. There were no differences in neurologic outcome between treatment and control groups. A subgroup analysis was performed for 84 neonates with acute perinatal brain injury (57 HIE, 18 stroke, 9 ICH), most of whom (70%) had neonatal seizures. There was a significant negative correlation between seizure burden and developmental scores (p<0.01). Associations between seizure burden and developmental scores were stronger in HIE and stroke groups compared with ICH (p<0.05). Conclusion: Bumetanide showed no long-term beneficial or adverse effects, as expected based on treatment duration versus duration of neonatal seizures. For neonates with perinatal brain injury, higher neonatal seizure burden correlated significantly with worse developmental outcome, particularly for ischemic versus hemorrhagic brain injury. These data highlight the need for further investigation of the long-term effects of both neonatal seizure severity and etiology.

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.

2-Photon imaging of fluorescent proteins in living swine.

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

A dynamic balance between neuronal death and clearance after acute brain injury.

After acute brain injury, neuronal apoptosis may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. 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 two 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 vital stains. 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 apoptotic neurons, and had either no or opposing effects on healthy surviving neurons. Significance: After 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 assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays. More accurate estimates of death rates will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

2-Photon imaging of fluorescent proteins in living swine.

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

Gene therapy for epilepsy.

On-demand inhibition of neuronal activity reduced spontaneous seizures in mice.

Clinical Reasoning: An 8-Year-Old With Acute Onset Ataxia.

Acute ataxia is a common neurologic presentation in the pediatric population that carries a broad differential diagnosis. The tempo of the presentation, distribution of the ataxia (focal or diffuse), examination findings, and paraclinical testing may be helpful in guiding diagnosis and management. Although Guillain-Barré syndrome (GBS) and its variant, Miller Fisher syndrome (MFS), are well defined, frequently encountered acute autoimmune neuropathies, the GBS/MFS spectrum have at least 12 different phenotypes with distinct neurologic features, 4 of which include ataxia. These lesser-known variants can be diagnosed clinically, in the absence of conclusive laboratory or neuroimaging data, and should always be considered in an acute presentation of ataxia. In this article, we present a previously healthy 8-year-old with acute onset ataxia with associated hyporeflexia that occurred after resolution of a presumed viral infection. We discuss our approach to ataxia, the patient's neurodiagnostic odyssey, and highlight the final diagnosis of acute ataxic neuropathy without ophthalmoplegia-a rare incomplete MFS subtype. Owing to timely recognition of the condition, the patient was treated appropriately and recovered fully.

Robust, long-term video EEG monitoring in a porcine model of post-traumatic epilepsy.

To date, post-traumatic epilepsy (PTE) research in large animal models has been limited. Recent advances in neocortical microscopy have made possible new insights into neocortical PTE. However, it is very difficult to engender convincing neocortical PTE in rodents. Thus, large animal models that develop neocortical PTE may provide useful insights that also can be more comparable to human patients. Because gyrencephalic species have prolonged latent periods, long-term video EEG recording is required. Here, we report a fully subcutaneous EEG implant with synchronized video in freely ambulatory swine for up to 13 months during epileptogenesis following bilateral cortical impact injuries or sham surgery The advantages of this system include the availability of a commercially available system that is simple to install, a low failure rate after surgery for EEG implantation, radiotelemetry that enables continuous monitoring of freely ambulating animals, excellent synchronization to video to EEG, and a robust signal to noise ratio. The disadvantages of this system in this species and age are the accretion of skull bone which entirely embedded a subset of skull screws and EEG electrodes, and the inability to rearrange the EEG electrode array. These disadvantages may be overcome by splicing a subdural electrode strip to the electrode leads so that skull growth is less likely to interfere with long-term signal capture and by placing two implants for a more extensive montage. This commercially available system in this bilateral cortical impact swine model may be useful to a wide range of investigators studying epileptogenesis in PTE.SignificancePost-traumatic epilepsy (PTE) is a cause of significant morbidity after traumatic brain injury (TBI) and is often drug-resistant. Robust, informative animal models would greatly facilitate PTE research. Ideally, this biofidelic model of PTE would utilize a species that approximates human brain anatomy, brain size, glial populations, and inflammatory pathways. An ideal model would also incorporate feasible methods for long-term video EEG recording required to quantify seizure activity. Here, we describe the first model of PTE in swine and describe a method for robust long-term video EEG monitoring for up to 13 months post-TBI. The relatively easy "out-of-the-box" radiotelemetry system and surgical techniques described here will be adaptable by a wide array of investigators studying the pathogenesis and treatment of PTE.

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.

Analysis of brain region-specific mRNA synthesis and stability by utilizing adult mouse brain slice culture.

Utilization of live animals for mechanistic study is challenging yet pivotal to elucidate pathogenesis of neurological diseases. Here, we present a protocol that employs cultured brain slices derived from adult mice to examine mRNA metabolism. We describe the preparation of acute brain slices and the treatments of RNA synthesis inhibitor and nucleotide analog to examine the effects of ataxin-1 loss-of-function on Bace1 mRNA stability and transcription in cortex. This protocol also includes electrophysiological recording of spontaneous neuronal activity in hippocampus. For complete details on the use and execution of this protocol, please refer to Suh et al. (2019).

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.

Risk and the Republican National Convention: Application of the Novel COVID-19 Operational Risk Assessment.

The United States Centers for Disease Control and Prevention and the World Health Organization broadly categorize mass gathering events as high risk for amplification of coronavirus disease 2019 (COVID-19) spread in a community due to the nature of respiratory diseases and the transmission dynamics. However, various measures and modifications can be put in place to limit or reduce the risk of further spread of COVID-19 for the mass gathering. During this pandemic, the Johns Hopkins University Center for Health Security produced a risk assessment and mitigation tool for decision-makers to assess SARS-CoV-2 transmission risks that may arise as organizations and businesses hold mass gatherings or increase business operations: The JHU Operational Toolkit for Businesses Considering Reopening or Expanding Operations in COVID-19 (Toolkit). This article describes the deployment of a data-informed, risk-reduction strategy that protects local communities, preserves local health-care capacity, and supports democratic processes through the safe execution of the Republican National Convention in Charlotte, North Carolina. The successful use of the Toolkit and the lessons learned from this experience are applicable in a wide range of public health settings, including school reopening, expansion of public services, and even resumption of health-care delivery.

Government response to COVID-19: Gaps revealed.

Drawing on the diverse perspectives of four emergency management professionals and a public administration academic, gaps revealed by our nation's response to the COVID-19 pandemic are discussed. These gaps range from political theory regarding our government system of federalism to fundamental questions around the public communication of risk management and the provision of mass shelter and care.

Antiepileptogenesis and disease modification: Progress, challenges, and the path forward-Report of the Preclinical Working Group of the 2018 NINDS-sponsored antiepileptogenesis and disease modification workshop.

Epilepsy is one of the most common chronic brain diseases and is often associated with cognitive, behavioral, or other medical conditions. The need for therapies that would prevent, ameliorate, or cure epilepsy and the attendant comorbidities is a priority for both epilepsy research and public health. In 2018, the National Institute of Neurological Disease and Stroke (NINDS) convened a workshop titled "Accelerating the Development of Therapies for Antiepileptogenesis and Disease Modification" that brought together preclinical and clinical investigators and industry and regulatory bodies' representatives to discuss and propose a roadmap to accelerate the development of antiepileptogenic (AEG) and disease-modifying (DM) new therapies. This report provides a summary of the discussions and proposals of the Preclinical Science working group. Highlights of the progress of collaborative preclinical research projects on AEG/DM of ongoing research initiatives aiming to improve infrastructure and translation to clinical trials are presented. Opportunities and challenges of preclinical epilepsy research, vis-à-vis clinical research, were extensively discussed, as they pertain to modeling of specific epilepsy types across etiologies and ages, the utilization of preclinical models in AG/DM studies, and the strategies and study designs, as well as on matters pertaining to transparency, data sharing, and reporting research findings. A set of suggestions on research initiatives, infrastructure, workshops, advocacy, and opportunities for expanding the borders of epilepsy research were discussed and proposed as useful initiatives that could help create a roadmap to accelerate and optimize preclinical translational AEG/DM epilepsy research.

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 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.

KCC2 Chloride Transport Contributes to the Termination of Ictal Epileptiform Activity.

Recurrent seizures intensely activate GABAA receptors (GABAA-Rs), which induces transient neuronal chloride ([Cl-]i) elevations and depolarizing GABA responses that contribute to the failure of inhibition that engenders further seizures and anticonvulsant resistance. The K+-Cl- cotransporter KCC2 is responsible for Cl- extrusion and restoration of [Cl-]i equilibrium (ECl) after synaptic activity, but at the cost of increased extracellular potassium which may retard K+-Cl- extrusion, depolarize neurons, and potentiate seizures. Thus, KCC2 may either diminish or facilitate seizure activity, and both proconvulsant and anticonvulsant effects of KCC2 inhibition have been reported. It is now necessary to identify the loci of these divergent responses by assaying both the electrographic effects and the ionic effects of KCC2 manipulation. We therefore determined the net effects of KCC2 transport activity on cytoplasmic chloride elevation and Cl- extrusion rates during spontaneous recurrent ictal-like epileptiform discharges (ILDs) in organotypic hippocampal slices in vitro, as well as the correlation between ionic and electrographic effects. We found that the KCC2 antagonist VU0463271 reduced Cl- extrusion rates, increased ictal [Cl-]i elevation, increased ILD duration, and induced status epilepticus (SE). In contrast, the putative KCC2 upregulator CLP257 improved chloride homeostasis and reduced the duration and frequency of ILDs in a concentration-dependent manner. Our results demonstrate that measuring both the ionic and electrographic effects of KCC2 transport clarify the impact of KCC2 modulation in specific models of epileptiform activity. Anticonvulsant effects predominate when KCC2-mediated chloride transport rather than potassium buffering is the rate-limiting step in restoring ECl and the efficacy of GABAergic inhibition during recurrent ILDs.