Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription.

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.

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

Intraventricular haemorrhage 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 intraventricular haemorrhage are biomarkers of hypoxic-ischaemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction in these conditions. This is because immature neurons express the chloride, salt and water transporter NKCC1, which subserves regulatory volume increases in non-neural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic-ischaemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters are diminished. Consequentially, mature neurons swell (cytotoxic oedema), whereas immature neurons might shrink. After hypoxic-ischaemic 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, in addition to prevention of neuronal shrinkage and the ensuing intraventricular haemorrhage, 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.

Timing of interventions to control neuronal chloride elevation in a model of neonatal seizures after hippocampal injury.

OBJECTIVE: Following hypoxic-ischemic (HI) brain injury, neuronal cytoplasmic chloride concentration ([Cl-]i) increases, potentially contributing to depolarizing γ-aminobutyric acid (GABA) responses, onset of seizures, and the failure of antiepileptic drugs that target inhibitory chloride-permeable GABAA receptors. Post-HI seizures characteristically begin hours after injury, by which time substantial accumulation of [Cl-]i may have already occurred. In immature neurons, a major pathway for Cl- influx is the reversible Na+-K+-2Cl- cotransporter NKCC1. METHODS: Spontaneous neuronal network, neuronal [Cl-]i, and GABA activity were determined in hippocampal preparations from neonatal Clomeleon and SuperClomeleon/DLX-cre mice to test whether blocking NKCC1 earlier after oxygen-glucose deprivation (OGD) injury would more effectively ameliorate the increase in [Cl-]i, ictallike epileptiform discharges (ILDs), and the failure of the GABAergic anticonvulsant phenobarbital. RESULTS: In vitro, murine intact hippocampi were free of ILDs for 12 h after preparation. Transient OGD resulted in a gradual increase in [Cl-]i, depolarizing action of GABA, and facilitation of neuronal network activity. Spontaneous ILDs began 3-5 h after injury. Blocking NKCC1 with 2-10 µmol·L-1 bumetanide reduced [Cl-]i equally well when applied up to 10 h after injury. Whereas phenobarbital or bumetanide applied separately were less effective when applied later after injury, ILDs were successfully suppressed by the combination of phenobarbital and bumetanide regardless of the number of prior ILDs or delay in application. SIGNIFICANCE: The present age-specific group studies demonstrate that after OGD, NKCC1 transport activity significantly contributes to progressive [Cl-]i accumulation, depolarizing action of GABA, and delayed onset of ILDs. In this neonatal model of neuronal injury and ILDs, earlier treatment with bumetanide alone more efficiently recovered control baseline [Cl-]i and depressed epileptiform discharges. However, there was no time dependency to the anti-ictal efficacy of the combination of phenobarbital and bumetanide. These in vitro results suggest that after perinatal injury, early pre-emptive treatment with phenobarbital plus bumetanide would be as efficacious as late treatment after seizures are manifest.

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.

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.

Traumatic alterations in GABA signaling disrupt hippocampal network activity in the developing brain.

Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl(-)](i)) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl(-)](i) correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl(-)](i) demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures.

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.

Interictal spikes, seizures and ictal cell death are not necessary for post-traumatic epileptogenesis in vitro.

Clinical studies indicate that phenytoin prevents acute post-traumatic seizures but not subsequent post-traumatic epilepsy. We explored this phenomenon using organotypic hippocampal slice cultures as a model of severe traumatic brain injury. Hippocampal slices were cultured for up to eight weeks, during which acute and chronic electrical recordings revealed a characteristic evolution of spontaneous epileptiform discharges, including interictal spikes, seizure activity and electrical status epilepticus. Cell death exhibited an early peak immediately following slicing, and a later secondary peak that coincided with the peak of seizure-like activity. The secondary peak in neuronal death was abolished by either blockade of glutamatergic transmission with kynurenic acid or by elimination of ictal activity and status epilepticus with phenytoin. Withdrawal of kynurenic acid or phenytoin was followed by a sharp increase in spontaneous seizure activity. Phenytoin's anticonvulsant and neuroprotective effects failed after four weeks of continuous administration. These data support the clinical findings that after brain injury, anticonvulsants prevent seizures but not epilepsy or the development of anticonvulsant resistance. We extend the clinical data by showing that secondary neuronal death is correlated with ictal but not interictal activity, and that blocking all three of these sequelae of brain injury does not prevent epileptogenesis in this in vitro model.

NKCC1 transporter facilitates seizures in the developing brain.

During development, activation of Cl(-)-permeable GABA(A) receptors (GABA(A)-R) excites neurons as a result of elevated intracellular Cl(-) levels and a depolarized Cl(-) equilibrium potential (E(Cl)). GABA becomes inhibitory as net outward neuronal transport of Cl(-) develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) facilitates the accumulation of Cl(-) in neurons. The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABA(A)-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

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).

Progressive NKCC1-dependent neuronal chloride accumulation during neonatal seizures.

Seizures induce excitatory shifts in the reversal potential for GABA(A)-receptor-mediated responses, which may contribute to the intractability of electro-encephalographic seizures and preclude the efficacy of widely used GABAergic anticonvulsants such as phenobarbital. We now report that, in intact hippocampi prepared from neonatal rats and transgenic mice expressing Clomeleon, recurrent seizures progressively increase the intracellular chloride concentration ([Cl(-)](i)) assayed by Clomeleon imaging and invert the net effect of GABA(A) receptor activation from inhibition to excitation assayed by the frequency of action potentials and intracellular Ca(2+) transients. These changes correlate with increasing frequency of seizure-like events and reduction in phenobarbital efficacy. The Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter blocker bumetanide inhibited seizure-induced neuronal Cl(-) accumulation and the consequent facilitation of recurrent seizures. Our results demonstrate a novel mechanism by which seizure activity leads to [Cl(-)](i) accumulation, thereby increasing the probability of subsequent seizures. This provides a potential mechanism for the early crescendo phase of neonatal seizures.

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.

Bumetanide enhances phenobarbital efficacy in a neonatal seizure model.

OBJECTIVES: High levels of expression of the Na+-K+-2Cl- (NKCC1) cotransporter in immature neurons cause the accumulation of intracellular chloride and, therefore, a depolarized Cl- equilibrium potential (E(Cl)). This results in the outward flux of Cl- through GABA(A) channels, the opposite direction compared with mature neurons, in which GABA(A) receptor activation is inhibitory because Cl- flows into the cell. This outward flow of Cl- in neonatal neurons is excitatory and contributes to a greater seizure propensity and poor electroencephalographic response to GABAergic anticonvulsants such as phenobarbital and benzodiazepines. Blocking the NKCC1 transporter with bumetanide prevents outward Cl- flux and causes a more negative GABA equilibrium potential (E(GABA)) in immature neurons. We therefore tested whether bumetanide enhances the anticonvulsant action of phenobarbital in the neonatal brain METHODS: Recurrent seizures were induced in the intact hippocampal preparation in vitro by continuous 5-hour exposure to low-Mg2+ solution. The anticonvulsant efficacy of phenobarbital, bumetanide, and the combination of these drugs was studied RESULTS: Phenobarbital failed to abolish or depress recurrent seizures in 70% of hippocampi. In contrast, phenobarbital in combination with bumetanide abolished seizures in 70% of hippocampi and significantly reduced the frequency, duration, and power of seizures in the remaining 30% INTERPRETATION: Thus, alteration of Cl- transport by bumetanide enables the anticonvulsant action of phenobarbital in immature brain. This is a mechanistic demonstration of rational anticonvulsant polypharmacy. The combination of these agents may comprise an effective therapy for early-life seizures.

Transient ischemia facilitates neuronal chloride accumulation and severity of seizures.

OBJECTIVE: Preceding oxygen glucose deprivation (OGD) and ongoing seizures have both been reported to increase neuronal chloride concentration ([Cl-]i), which may contribute to anticonvulsant failure by reversing the direction of chloride currents at inhibitory GABAA synapses. METHODS: The effects of OGD on [Cl-]i, seizure activity, and anticonvulsant efficacy were studied in a chronically epileptic in vitro preparation. RESULTS: Seizures initially increased during OGD, followed by suppression. On reperfusion, seizure frequency and [Cl-]i progressively increased, and phenobarbital efficacy was reduced. Bumetanide (10 µmol/L) and furosemide (1 mmol/L) prevented or reduced the OGD induced [Cl-]i increase. Phenobarbital efficacy was enhanced by bumetanide (10 µmol/L). Furosemide (1 mmol/L) suppressed recurrent seizures. INTERPRETATION: [Cl-]i increases after OGD and is associated with worsened seizure activity, reduced efficacy of GABAergic anticonvulsants, and amelioration by antagonists of secondary chloride transport.

Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential.

Pharmacoresistant seizures and cytotoxic cerebral edema are serious complications of ischemic and traumatic brain injury. Intraneuronal Cl- concentration ([Cl-]i) regulation impacts on both cell volume homeostasis and Cl--permeable GABAA receptor-dependent membrane excitability. Understanding the pleiotropic molecular determinants of neuronal [Cl-]i - cytoplasmic impermeant anions, polyanionic extracellular matrix (ECM) glycoproteins, and plasmalemmal Cl- transporters - could help the identification of novel anticonvulsive and neuroprotective targets. The cation/Cl- cotransporters and ECM metalloproteinases may be particularly druggable targets for intervention. We establish here a paradigm that accounts for recent data regarding the complex regulatory mechanisms of neuronal [Cl-]i and how these mechanisms impact on neuronal volume and excitability. We propose approaches to modulate [Cl-]i that are relevant for two common clinical sequela of brain injury: edema and seizures.

Excitatory actions of endogenously released GABA contribute to initiation of ictal epileptiform activity in the developing hippocampus.

In the developing rat hippocampus, ictal epileptiform activity can be elicited easily in vitro during the first three postnatal weeks. Changes in neuronal ion transport during this time cause the effects of GABA(A) receptor (GABA(A)-R) activation to shift gradually from strongly depolarizing to hyperpolarizing. It is not known whether the depolarizing effects of GABA and the propensity for ictal activity are causally linked. A key question is whether the GABA-mediated depolarization is excitatory, which we defined operationally as being sufficient to trigger action potentials. We assessed the effect of endogenous GABA on ictal activity and neuronal firing rate in hippocampal slices from postnatal day 1 (P1) to P30. In extracellular recordings, there was a strong correlation between the postnatal age at which GABA(A)-R antagonists decreased action potential frequency (P23) and the age at which ictal activity could be induced by elevated potassium (P23). In addition, there was a strong correlation between the fraction of slices in which ictal activity was induced by elevated potassium concentrations and the fractional decrease in action potential firing when GABA(A)-Rs were blocked in the presence of ionotropic glutamate receptor antagonists. Finally, ictal activity induced by elevated potassium was blocked by the GABA(A)-R antagonists bicuculline and SR-95531 (gabazine) and increased in frequency and duration by GABA(A)-R agonists isoguvacine and muscimol. Thus, the propensity of the developing hippocampus for ictal activity is highly correlated with the effect of GABA on action potential probability and reversed by GABA(A) antagonists, indicating that GABA-mediated excitation is causally linked to ictal activity in this developmental window.

Mechanisms of fast ripples in the hippocampus.

Hippocampal fast ripples (FRs) have been associated with seizure onset in both human and experimental epilepsy. To characterize the mechanisms underlying FR oscillations (200-600 Hz), we studied activity of single neurons and neuronal networks in rat hippocampal slices in vitro. The correlation between the action potentials of bursting pyramidal cells and local field potential oscillations suggests that synchronous onset of action potential bursts and similar intrinsic firing patterns among local neurons are both necessary conditions for FR oscillations. Increasing the fidelity of individual pyramidal cell spike train timing by blocking accommodation dramatically increased FR amplitude, whereas blockade of potassium conductances decreased the fidelity of action potential timing in individual pyramidal cell action potential bursts and decreased FR amplitude. Blockade of ionotropic glutamate receptors desynchronized onset of action potential bursts in individual pyramidal cells and abolished fast ripples. Thus, synchronous burst onset mediated by recurrent excitatory synaptic transmission and similar intrinsic spike timing mechanisms in neighboring pyramidal cells are necessary conditions for FR oscillations within the hippocampal network.

Transition from interictal to ictal activity in limbic networks in vitro.

The transition from brief bursts of synchronous population activity characteristic of interictal epileptiform discharges (IEDs) to more prolonged epochs of population activity characteristic of seizures (ictal-like activity) was recorded in juvenile rat hippocampal-entorhinal cortex slices and hippocampal slices using multiple-site extracellular electrodes. Epileptiform activity was elicited by either increased extracellular potassium or 4-AP. IEDs originated in the CA3 a-b region and spread bidirectionally into CA1 and CA3c dentate gyrus. The transition from IEDs to ictal-like sustained epileptiform activity was reliably preceded by (1) increase in IED propagation velocity, (2) increase in IED secondary afterdischarges and their reverberation between CA3a and CA3c, and (3) shift in the IED initiation area from CA3 a-b to CA3c. Ictal-like sustained network oscillations (10-20 Hz) originated in CA3c and spread to CA1. The pattern of hippocampal ictal-like activity was unaffected by removal of the entorhinal cortex. These findings indicate that interictal and ictal activity can originate in the same neural network, and that the transition from interictal to ictal-like-sustained activity is preceded by predictable alterations in the origin and spread of IEDs. These findings elucidate new targets for investigating the proximate causes, prediction, and treatment of seizures.

Acute and chronic efficacy of bumetanide in an in vitro model of posttraumatic epileptogenesis.

BACKGROUND: Seizures triggered by acute injuries to the developing brain respond poorly to first-line medications that target the inhibitory chloride-permeable GABAA receptor. Neuronal injury is associated with profound increases in cytoplasmic chloride ([Cl(-)]i) resulting in depolarizing GABA signaling, higher seizure propensity and limited efficacy of GABAergic anticonvulsants. The Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter blocker bumetanide reduces [Cl(-)]i and causes more negative GABA equilibrium potential in injured neurons. We therefore tested both the acute and chronic efficacy of bumetanide on early posttraumatic ictal-like epileptiform discharges and epileptogenesis. METHODS: Acute hippocampal slices were used as a model of severe traumatic brain injury and posttraumatic epileptogenesis. Hippocampal slices were then incubated for 3 weeks. After a 1-week latent period, slice cultures developed chronic spontaneous ictal-like discharges. The anticonvulsant and anti-epileptogenic efficacy of bumetanide, phenobarbital, and the combination of these drugs was studied. RESULTS: Bumetanide reduced the frequency and power of early posttraumatic ictal-like discharges in vitro and enhanced the anticonvulsant efficacy of phenobarbital. Continuous 2-3 weeks administration of bumetanide as well as phenobarbital in combination with bumetanide failed to prevent posttraumatic ictal-like discharges and epileptogenesis. CONCLUSIONS: Our data demonstrate a persistent contribution of NKCC1 cotransport in posttraumatic ictal-like activity, presumably as a consequence of chronic alterations in neuronal chloride homeostasis and GABA-mediated inhibition. New strategies for more effective reduction in posttraumatic and seizure-induced [Cl(-)]i accumulation could provide the basis for effective treatments for posttraumatic epileptogenesis and the resultant seizures.