Increased propensity for infantile spasms and altered neocortical excitation-inhibition balance in a mouse model of down syndrome carrying human chromosome 21.

Children with Down syndrome (DS, trisomy of chromosome 21) have an increased risk of infantile spasms (IS). As an epileptic encephalopathy, IS may further impair cognitive function and exacerbate neurodevelopmental delays already present in children with DS. To investigate the pathophysiology of IS in DS, we induced IS-like epileptic spasms in a genetic mouse model of DS that carries human chromosome 21q, TcMAC21, the animal model most closely representing gene dosage imbalance in DS. Repetitive extensor/flexor spasms were induced by the GABAB receptor agonist γ-butyrolactone (GBL) and occurred predominantly in young TcMAC21 mice (85%) but also in some euploid mice (25%). During GBL application, background electroencephalographic (EEG) amplitude was reduced, and rhythmic, sharp-and-slow wave activity or high-amplitude burst (epileptiform) events emerged in both TcMAC21 and euploid mice. Spasms occurred only during EEG bursts, but not every burst was accompanied by a spasm. Electrophysiological experiments revealed that basic membrane properties (resting membrane potential, input resistance, action-potential threshold and amplitude, rheobase, input-output relationship) of layer V pyramidal neurons were not different between TcMAC21 mice and euploid controls. However, excitatory postsynaptic currents (EPSCs) evoked at various intensities were significantly larger in TcMAC21 mice than euploid controls, while inhibitory postsynaptic currents (IPSCs) were similar between the two groups, resulting in an increased excitation-inhibition (E-I) ratio. These data show that behavioral spasms with epileptic EEG activity can be induced in young TcMAC21 DS mice, providing proof-of-concept evidence for increased IS susceptibility in these DS mice. Our findings also show that basic membrane properties are similar in TcMAC21 and euploid mice, while the neocortical E-I balance is altered to favor increased excitation in TcMAC21 mice, which may predispose to IS generation.

Antiseizure effect of 2-deoxyglucose is not dependent on the presynaptic vacuole ATP pump or the somatic ATP-sensitive K+ channel.

Inhibition of glycolysis with 2-deoxyglucose (2-DG) produces antiseizure effects in brain slices and animal models, yet the mechanisms remain elusive. Here, we examined two glycolysis-derived ATP-associated mechanisms: vacuole ATP pump (V-ATPase) and ATP-sensitive K+ channel (KATP). Epileptiform bursts were generated in the CA3 area of hippocampal slices by 0 Mg2+ and 4-aminopyridine. 2-DG consistently abolished epileptiform bursts in the presence of pyruvate (to sustain tricarboxylic acid cycle for oxidative ATP production) at 30-33°C but not at room temperature (22°C). Under physiological conditions, 2-DG did not reduce the amplitude of evoked excitatory postsynaptic currents (EPSCs) or the paired-pulse ratio in CA3 neurons. During repetitive high-frequency (20 Hz, 20-50 pulses) stimulation, 2-DG did not accelerate the decline of EPSCs (i.e., depletion of transmitter release), even when preincubated with 8 mM K+ to enhance activity-dependent uptake of 2-DG. In addition, in 2-DG tetanic stimulation (200 Hz, 1 s) dramatically increased rather than diminished the occurrence of spontaneous EPSCs immediately after stimulation (i.e., no transmitter depletion). Moreover, a V-ATPase blocker (concanamycin) failed to block epileptiform bursts that were subsequently abolished by 2-DG. Furthermore, 2-DG did not induce detectable KATP current in hippocampal neurons. Finally, epileptiform bursts were not affected by either a KATP opener (diazoxide) or a KATP blocker (glibenclamide) but were blocked by 2-DG in the same slices. Altogether, these data suggest that 2-DG's antiseizure action is temperature dependent and achieved exclusively by inhibition of glycolysis and is not likely to be mediated by the two membrane-bound ATP-associated machinery mechanisms, V-ATPase and KATP.NEW & NOTEWORTHY Inhibition of glycolysis with 2-deoxyglucose (2-DG) represents a novel metabolic antiseizure approach, yet the mechanisms remain elusive. Here, we show that 2-DG's antiseizure action is both glycolysis and temperature dependent but not mediated by the vacuole ATP pump (V-ATPase) or ATP-sensitive K+ channel (KATP). Our data provide new insights to understand 2-DG's cellular mechanisms of action and, more broadly, neuronal metabolism and excitability.

Enhanced burst discharges in the CA1 area of the immature versus adult hippocampus: patterns and cellular mechanisms.

Burst discharges in the immature brain may contribute to its enhanced seizure susceptibility. The cellular mechanisms underlying burst discharges in the CA1 area of the immature versus adult hippocampus were investigated with simultaneous whole-cell and field-potential recordings. When GABAA receptors were blocked pharmacologically, bursts in CA1 were either graded or all-or-none (or mixed) as a function of electrical stimulation intensity. Most CA1 minislices from immature rats displayed all-or-none or mixed bursts, whereas the slices from adult rats predominantly elicited graded bursts. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were greater in CA1 pyramidal cells from the immature than the adult slices. The developmental differences in CA1 bursting were also detected in slices adjusted for maturational changes in brain volume (i.e., 350 µm thick for immature vs. 450 µm thick for adult rats). Neither N-methyl-d-aspartate (NMDA) nor group I metabotropic glutamate (mGlu1) receptor antagonists blocked the network-driven bursts in immature CA1, but an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker abolished them. Robust excitatory postsynaptic potentials (EPSPs) occurred after bursts in some immature CA1 slices (23%) but never in slices from the adult. The input-output (amount of current injected vs. number of action potentials generated) relationship was markedly greater in CA1 pyramidal cells in the immature compared with the adult hippocampus. These data suggest that the CA1 area of the immature brain is capable of generating network-driven bursts, which declines in adult rats. The increased propensity of burst generation in immature CA1 appears to involve a greater AMPA receptor-mediated synaptic network and an increased intrinsic spike-generating ability.NEW & NOTEWORTHY Burst discharges in the developing brain can provide valuable insights into epileptogenesis. We show that the immature hippocampal CA1 area is capable of generating all-or-none (i.e., network) bursts, which transitions to graded (i.e., nonnetwork) bursts in the mature brain via both synaptic and intrinsic mechanisms. Our results provide new clues to help understand possible mechanisms that may be shared in the immature and epileptic brain and how the normal brain becomes seizure prone (i.e., epileptogenesis).

2-deoxyglucose and ß-hydroxybutyrate fail to attenuate seizures in the betamethasone-NMDA model of infantile spasms.

Infantile spasms (IS) is an epileptic encephalopathy with a poor neurodevelopmental prognosis, and limited, often ineffective treatment options. The effectiveness of metabolic approaches to seizure control is being increasingly shown in a wide variety of epilepsies. This study investigates the efficacy of the glycolysis inhibitor 2-deoxyglucose (2-DG) and the ketone body ß-hydroxybutyrate (BHB) in the betamethasone-NMDA model of rat IS. Prenatal rats were exposed to betamethasone on gestational day 15 (G15) and NMDA on postnatal day 15 (P15). Video-electroencephalography (v-EEG) was used to monitor spasms. NMDA consistently induced hyperflexion spasms associated with interictal sharp-slow wave EEG activity and ictal flattening of EEG signals, reminiscent of hypsarrhythmia and electrodecrement, respectively. 2-DG (500 mg/kg, i.p), BHB (200 mg/kg, i.p.), or both were administered immediately after occurrence of the first spasm. No experimental treatment altered significantly the number, severity, or progression of spasms compared with saline treatment. These data suggest that metabolic inhibition of glycolysis or ketogenesis does not reduce infantile spasms in the NMDA model. The study further validates the betamethasone-NMDA model in terms of its behavioral and electrographic resemblance to human IS and supports its use for preclinical drug screening.

Targeting ischemia-induced KCC2 hypofunction rescues refractory neonatal seizures and mitigates epileptogenesis in a mouse model.

Neonatal seizures pose a clinical challenge in their early detection, acute management, and long-term comorbidities. They are often caused by hypoxic-ischemic encephalopathy and are frequently refractory to the first-line antiseizure medication phenobarbital. One proposed mechanism for phenobarbital inefficacy during neonatal seizures is the reduced abundance and function of the neuron-specific K+/Cl− cotransporter 2 (KCC2), which maintains chloride homeostasis and promotes GABAergic inhibition upon its phosphorylation during postnatal development. Here, we investigated whether this mechanism is causal and whether it can be rescued by KCC2 functional enhancement. In a CD-1 mouse model of refractory ischemic neonatal seizures, treatment with the KCC2 functional enhancer CLP290 rescued phenobarbital efficacy, increased KCC2 abundance, and prevented the development of epileptogenesis, as quantified by video electroencephalogram monitoring. These effects were prevented by knock-in expression of nonphosphorylatable mutants of KCC2 (S940A or T906A and T1007A), indicating that KCC2 phosphorylation regulates both neonatal seizure susceptibility and CLP290-mediated KCC2 functional enhancement. Our findings therefore validate KCC2 as a clinically relevant target for refractory neonatal seizures and provide insights for future drug development.

Na+-K+-ATPase functions in the developing hippocampus: regional differences in CA1 and CA3 neuronal excitability and role in epileptiform network bursting.

The Na+-K+-ATPase (Na+-K+ pump) is essential for setting resting membrane potential and restoring transmembrane Na+ and K+ gradients after neuronal firing, yet its roles in developing neurons are not well understood. This study examined the contribution of the Na+-K+ pump to resting membrane potential and membrane excitability of developing CA1 and CA3 neurons and its role in maintaining synchronous network bursting. Experiments were conducted in postnatal day (P)9 to P13 rat hippocampal slices using whole cell patch-clamp and extracellular field-potential recordings. Blockade of the Na+-K+ pump with strophanthidin caused marked depolarization (23.1 mV) in CA3 neurons but only a modest depolarization (3.3 mV) in CA1 neurons. Regarding other membrane properties, strophanthidin differentially altered the voltage-current responses, input resistance, action-potential threshold and amplitude, rheobase, and input-output relationship in CA3 vs. CA1 neurons. At the network level, strophanthidin stopped synchronous epileptiform bursting in CA3 induced by 0 Mg2+ and 4-aminopyridine. Furthermore, dual whole cell recordings revealed that strophanthidin disrupted the synchrony of CA3 neuronal firing. Finally, strophanthidin reduced spontaneous excitatory postsynaptic current (sEPSC) bursts (i.e., synchronous transmitter release) and transformed them into individual sEPSC events (i.e., nonsynchronous transmitter release). These data suggest that the Na+-K+ pump plays a more profound role in membrane excitability in developing CA3 neurons than in CA1 neurons and that the pump is essential for the maintenance of synchronous network bursting in CA3. Compromised Na+-K+ pump function leads to cessation of ongoing synchronous network activity, by desynchronizing neuronal firing and neurotransmitter release in the CA3 synaptic network. These findings have implications for the regulation of network excitability and seizure generation in the developing brain.NEW & NOTEWORTHY Despite the extensive literature showing the importance of the Na+-K+ pump in various neuronal functions, its roles in the developing brain are not well understood. This study reveals that the Na+-K+ pump differentially regulates the excitability of CA3 and CA1 neurons in the developing hippocampus, and the pump activity is crucial for maintaining network activity. Compromised Na+-K+ pump activity desynchronizes neuronal firing and transmitter release, leading to cessation of ongoing epileptiform network bursting.

The efficacy of fructose-1,6-bisphosphate in suppressing status epilepticus in developing rats.

PURPOSE: Treatment of pediatric status epilepticus (SE) remains challenging as up to 50 % of patients are refractory to conventional anti-seizure medications. The glycolytic intermediate, fructose-1,6-bisphosphate (FBP), has been reported to exert significant anticonvulsant effects in both adult animals and in in vitro models of seizures. This study aims to examine FBP efficacy in controlling seizures in a rat model of juvenile SE. METHODS: Sprague Dawley rats (P11-P17) were injected with pilocarpine (300 mg/kg, i.p.) to induce SE, which was monitored by video-electroencephalography (v-EEG). Thirty minutes into SE, FBP was administrated (500 or 1000 mg/kg, i.p.). v-EEG recording was continued for ∼60 additional minutes to assess the anticonvulsant effect of FBP, compared with vehicle (saline) treatment. RESULTS: SE consistently occurred in rat pups 10-15 min after pilocarpine injection and persisted over the 90-min recording period. Neither saline nor a lower dose of FBP (500 mg/kg) treatment stopped behavioral and electrographic seizures. At higher doses (1000 mg/kg), FBP terminated SE in ∼15 min in 60 % (6 of 10) of the rat pups. CONCLUSION: The endogenous glycolytic metabolite, FBP, promptly suppresses ongoing seizure activity and represents a potential alternative metabolic therapy to improve the treatment of SE in the juvenile age range.

2-Deoxyglucose terminates pilocarpine-induced status epilepticus in neonatal rats.

OBJECTIVE: Neonatal status epilepticus (SE) is a life-threatening medical emergency. Unfortunately, up to 50% of neonates with SE are resistant to current antiseizure drugs, highlighting the need for better treatments. This study aims to explore a novel metabolic approach as a potential alternative treatment to control neonatal SE, using the glycolytic inhibitor 2-deoxyglucose (2-DG). METHODS: SE was induced by pilocarpine (300 mg/kg, intraperitoneally [ip]) in neonatal Sprague Dawley rats (postnatal day 10 [P10]-P17) and was monitored by video-electroencephalography (V-EEG). After 30 minutes of SE, 2-DG or one of two conventional antiseizure drugs with different mechanisms of action, phenobarbital or levetiracetam, was administrated ip, and V-EEG recording was continued for ~60 additional minutes. The time to seizure cessation after drug injection, EEG scores, and power spectra before and after drug or saline treatment were used to assess drug effects. RESULTS: Once SE became sustained, administration of 2-DG (50, 100, or 500 mg/kg, ip) consistently stopped behavioral and electrographic seizures within 10-15 minutes; lower doses took longer (25-30 minutes) to stop SE, demonstrating a dose-dependent effect. Administration of phenobarbital (30 mg/kg, ip) or levetiracetam (100 mg/kg, ip) also stopped SE within 10-15 minutes in neonatal rats. SIGNIFICANCE: Our results suggest that the glycolysis inhibitor 2-DG acts quickly to reduce neuronal hyperexcitability and effectively suppress ongoing seizure activity, which may provide translational value in the treatment of neonatal SE.

Infantile Spasms: An Update on Pre-Clinical Models and EEG Mechanisms.

Infantile spasms (IS) is an epileptic encephalopathy with unique clinical and electrographic features, which affects children in the middle of the first year of life. The pathophysiology of IS remains incompletely understood, despite the heterogeneity of IS etiologies, more than 200 of which are known. In particular, the neurobiological basis of why multiple etiologies converge to a relatively similar clinical presentation has defied explanation. Treatment options for this form of epilepsy, which has been described as "catastrophic" because of the poor cognitive, developmental, and epileptic prognosis, are limited and not fully effective. Until the pathophysiology of IS is better clarified, novel treatments will not be forthcoming, and preclinical (animal) models are essential for advancing this knowledge. Here, we review preclinical IS models, update information regarding already existing models, describe some novel models, and discuss exciting new data that promises to advance understanding of the cellular mechanisms underlying the specific EEG changes seen in IS-interictal hypsarrhythmia and ictal electrodecrement.

2-Deoxyglucose and Beta-Hydroxybutyrate: Metabolic Agents for Seizure Control.

Current anti-seizure drugs (ASDs) are believed to reduce neuronal excitability through modulation of ion channels and transporters that regulate excitability at the synaptic level. While most patients with epilepsy respond to ASDs, many remain refractory to medical treatment but respond favorably to a high-fat, low-carbohydrate metabolism-based therapy known as the ketogenic diet (KD). The clinical effectiveness of the KD has increasingly underscored the thesis that metabolic factors also play a crucial role in the dampening neuronal hyperexcitability that is a hallmark feature of epilepsy. This notion is further amplified by the clinical utility of other related metabolism-based diets such as the modified Atkins diet and the low-glycemic index treatment (LGIT). Traditional high-fat diets are characterized by enhanced fatty acid oxidation (which produces ketone bodies such as beta-hydroxybutyrate) and a reduction in glycolytic flux, whereas the LGIT is predicated mainly on the latter observation of reduced blood glucose levels. As dietary implementation is not without challenges regarding clinical administration and patient compliance, there is an inherent desire and need to determine whether specific metabolic substrates and/or enzymes might afford similar clinical benefits, hence validating the concept of a "diet in a pill." Here, we discuss the evidence for one glycolytic inhibitor, 2-deoxyglucose (2DG) and one metabolic substrate, ß-hydroxybutyrate (BHB) exerting direct effects on neuronal excitability, highlight their mechanistic differences, and provide the strengthening scientific rationale for their individual or possibly combined use in the clinical arena of seizure management.

Pediatric Epilepsy Mechanisms: Expanding the Paradigm of Excitation/Inhibition Imbalance.

Mechanisms underlying seizures and epilepsy have traditionally been considered to involve abnormalities of ion channels or synaptic function. Those considerations gave rise to the excitation/inhibition (E/I) imbalance theory, whereby increased excitation, decreased inhibition, or both favor a hyperexcitable state and an increased propensity for seizure generation and epileptogenesis. Several recent findings warrant reconsideration and expansion of the E/I hypothesis: novel genetic mutations have been identified that do not overtly affect E/I balance; neurotransmitters may exert paradoxical effects, especially during development; anti-seizure medications do not necessarily work by decreasing excitation or increasing inhibition; and metabolic factors participate in the regulation of neuronal and network excitability. These novel conceptual and experimental advances mandate expansion of the E/I paradigm, with the expectation that new and exciting therapies will emerge from this broadened understanding of how seizures and epilepsy arise and progress.

Glycolytic inhibition: A novel approach toward controlling neuronal excitability and seizures.

Conventional antiseizure medications reduce neuronal excitability through effects on ion channels or synaptic function. In recent years, it has become clear that metabolic factors also play a crucial role in the modulation of neuronal excitability. Indeed, metabolic regulation of neuronal excitability is pivotal in seizure pathogenesis and control. The clinical effectiveness of a variety of metabolism-based diets, especially for children with medication-refractory epilepsy, underscores the applicability of metabolic approaches to the control of seizures and epilepsy. Such diets include the ketogenic diet, the modified Atkins diet, and the low-glycemic index treatment (among others). A promising avenue to alter cellular metabolism, and hence excitability, is by partial inhibition of glycolysis, which has been shown to reduce seizure susceptibility in a variety of animal models as well as in cellular systems in vitro. One such glycolytic inhibitor, 2-deoxy-d-glucose (2DG), increases seizure threshold in vivo and reduces interictal and ictal epileptiform discharges in hippocampal slices. Here, we review the role of glucose metabolism and glycolysis on neuronal excitability, with specific reference to 2DG, and discuss the potential use of 2DG and similar agents in the clinical arena for seizure management.

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices.

Manipulation of metabolic pathways (e.g., ketogenic diet (KD), glycolytic inhibition) alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a "branching" metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP) in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP). Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa) in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5-5 mM) blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min), suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531), nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA). In contrast, F1,6BP caused moderate reduction (~50%) in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 µM), suggesting that these bursts are calcium dependent. Altogether, these data demonstrate that the glycolytic metabolite, F1,6BP, blocks epileptiform activity via a previously unrecognized extracellular effect on ICa, which provides new insight into the metabolic control of neural excitability.

Glycolytic inhibition by 2-deoxy-d-glucose abolishes both neuronal and network bursts in an in vitro seizure model.

Neuronal activity is energy demanding and coupled to cellular metabolism. In this study, we investigated the effects of glycolytic inhibition with 2-deoxy-d-glucose (2-DG) on basal membrane properties, spontaneous neuronal firing, and epileptiform network bursts in hippocampal slices. The effect of glycolytic inhibition on basal membrane properties was examined in hippocampal CA1 neurons, which are not ordinarily active spontaneously. Intracellular application of 2-DG did not significantly alter the membrane input resistance, action-potential threshold, firing pattern, or input-output relationship of these neurons compared with simultaneously recorded neighboring neurons without intracellular 2-DG. The effect of glycolytic inhibition on neuronal firing was tested in spontaneously active hippocampal neurons (CA3) when synaptic transmission was left intact or blocked with AMPA, NMDA, and GABAA receptor antagonists (DNQX, APV, and bicuculline, respectively). Under both conditions (synaptic activity intact or blocked), bath application of 2-DG (2 mM) blocked spontaneous firing in ~2/3 (67 and 71%, respectively) of CA3 pyramidal neurons. In contrast, neuronal firing of CA3 neurons persisted when 2-DG was applied intracellularly, suggesting that glycolytic inhibition of individual neurons is not sufficient to stop neuronal firing. The effects of 2-DG on epileptiform network bursts in area CA3 were tested in Mg2+-free medium containing 50 µM 4-aminopyridine. Bath application of 2-DG abolished these epileptiform bursts in a dose-dependent and all-or-none manner. Taken together, these data suggest that altered glucose metabolism profoundly affects cellular and network hyperexcitability and that glycolytic inhibition by 2-DG can effectively abrogate epileptiform activity.NEW & NOTEWORTHY Neuronal activity is highly energy demanding and coupled to cellular metabolism. In this study, we demonstrate that glycolytic inhibition with 2-deoxy-d-glucose (2-DG) effectively suppresses spontaneous neuronal firing and epileptiform bursts in hippocampal slices. These data suggest that an altered metabolic state can profoundly affect cellular and network excitability, and that the glycolytic inhibitor 2-DG may hold promise as a novel treatment of drug-resistant epilepsy.

Pediatric Epileptic Encephalopathies: Pathophysiology and Animal Models.

Epileptic encephalopathies are syndromes in which seizures or interictal epileptiform activity contribute to or exacerbate brain function, beyond that caused by the underlying pathology. These severe epilepsies begin early in life, are associated with poor lifelong outcome, and are resistant to most treatments. Therefore, they represent an immense challenge for families and the medical care system. Furthermore, the pathogenic mechanisms underlying the epileptic encephalopathies are poorly understood, hampering attempts to devise novel treatments. This article reviews animal models of the three classic epileptic encephalopathies-West syndrome (infantile spasms), Lennox-Gastaut syndrome, and continuous spike waves during sleep or Landau-Kleffner syndrome-with discussion of how animal models are revealing underlying pathophysiological mechanisms that might be amenable to targeted therapy.

Synaptic and extrasynaptic plasticity in glutamatergic circuits involving dentate granule cells following chronic N-methyl-D-aspartate receptor inhibition.

Chronic global N-methyl-d-aspartate receptor (NMDAR) blockade leads to changes in glutamatergic transmission. The impact of more subunit-selective NMDAR inhibition on glutamatergic circuits remains incomplete. To this end, organotypic hippocampal slice cultures were treated for 17-21 days with the high-affinity competitive antagonist d-aminophosphonovaleric acid (d-APV), the allosteric GluN2B-selective antagonist Ro25-6981, or the newer competitive GluN2A-preferring antagonist NVP-AAM077. Electrophysiological recordings from dentate granule cells revealed that chronic d-APV treatment increased, whereas chronic Ro25-6981 reduced, epileptiform event-associated large-amplitude spontaneous excitatory postsynaptic currents (sEPSC) compared with all other treatment groups, consistent with opposite effects on glutamatergic networks. Presynaptically, chronic d-APV or Ro25-6981 increased small-amplitude sEPSCs and AMPA/kainate receptor-mediated miniature EPSCs (mEPSCAMPAR) frequency. Chronic d-APV or NVP-AAM077, but not Ro25-6981, increased putative vGlut1-positive glutamatergic synapses. Postsynaptically, chronic d-APV dramatically increased mEPSCAMPAR and profoundly decreased NMDAR-mediated mEPSC (mEPSCNMDAR) measures, suggesting increased AMPAR/NMDAR ratio. Ro25-6981 decreased mEPSCAMPAR charge transfer and modestly decreased mEPSCNMDAR frequency and decay, suggesting downward scaling of AMPAR and NMDAR function without dramatically altering AMPAR/NMDAR ratio. Extrasynaptically, threo-ß-benzyloxyaspartate-enhanced "tonic" NMDAR current amplitude and activated channel number estimates were significantly increased only by chronic Ro25-6981. For intrinsic excitability, action potential threshold was slightly more negative following chronic d-APV or NVP-AAM077. The predominant pro-excitatory effects of chronic d-APV are consistent with increased glutamatergic transmission and network excitability. The minor effects of chronic NVP-AAM077 on action potential threshold and synapse number are consistent with minimal effects on circuit function. The chronic Ro25-6981-induced downward scaling of synaptic AMPAR and NMDAR function is consistent with decreased postsynaptic glutamate receptors and reduced network excitability.

Increased Kv1 channel expression may contribute to decreased sIPSC frequency following chronic inhibition of NR2B-containing NMDAR.

Numerous studies have documented the effects of chronic N-methyl-D-aspartate receptor (NMDAR) blockade on excitatory circuits, but the effects on inhibitory circuitry are not well studied. NR2A- and NR2B-containing NMDARs play differential roles in physiological processes, but the consequences of chronic NR2A- or NR2B-containing NMDAR inhibition on glutamatergic and GABAergic neurotransmission are unknown. We investigated altered GABAergic neurotransmission in dentate granule cells and interneurons following chronic treatment with the NR2B-selective antagonist, Ro25,6981, the NR2A-prefering antagonist, NVP-AAM077, or the non-subunit-selective NMDAR antagonist, D-APV, in organotypic hippocampal slice cultures. Electrophysiological recordings revealed large reductions in spontaneous inhibitory postsynaptic current (sIPSC) frequency in both granule cells and interneurons following chronic Ro25,6981 treatment, which was associated with minimally altered sIPSC amplitude, miniature inhibitory postsynaptic current (mIPSC) frequency, and mIPSC amplitude, suggesting diminished action potential-dependent GABA release. Chronic NVP-AAM077 or D-APV treatment had little effect on these measures. Reduced sIPSC frequency did not arise from downregulated GABA(A)R, altered excitatory or inhibitory drive to interneurons, altered interneuron membrane properties, increased failure rate, decreased action potential-dependent release probability, or mGluR/GABA(B) receptor modulation of GABA release. However, chronic Ro25,6981-mediated reductions in sIPSC frequency were occluded by the K+ channel blockers, dendrotoxin, margatoxin, and agitoxin, but not dendrotoxin-K or XE991. Immunohistochemistry also showed increased Kv1.2, Kv1.3, and Kv1.6 in the dentate molecular layer following chronic Ro25,6981 treatment. Our findings suggest that increased Kv1 channel expression/function contributed to diminished action potential-dependent GABA release following chronic NR2B-containing NMDAR inhibition and that these Kv1 channels may be heteromeric complexes containing Kv1.2, Kv1.3, and Kv1.6.

Repetitive perforant-path stimulation induces epileptiform bursts in minislices of dentate gyrus from rats with kainate-induced epilepsy.

The epileptic hippocampus has an enhanced propensity for seizure generation, but how spontaneous seizures start is poorly understood. Using whole cell and field-potential recordings, this study explored whether repetitive perforant-path stimulation at physiological frequencies could induce epileptiform bursts in dentate gyrus minislices from rats with kainate-induced epilepsy. Control slices from saline-treated rats responded to single perforant-path stimulation with an excitatory postsynaptic potential (EPSP) and a single population spike in normal medium, and repetitive stimulation at different frequencies (0.1, 1, 2, 5, 10 Hz) did not cause significant increases in the responses. Most minislices (82%) from rats with kainate-induced epilepsy also responded to single perforant-path stimulation with an EPSP and a single population spike/action potential, but some slices (18%) had a more robust response with a prolonged duration and negative DC shift or responses with two to three population spikes. Repetitive perforant-path stimulation at 5-10 Hz, however, transformed the single-spike responses into epileptiform bursts with multiple spikes in half (52%) of the slices, while lower frequency (e.g., ≤ 1 Hz) stimulation failed to produce these changes. The emergence of epileptiform bursts was consistently associated with a negative field-potential DC shift and membrane depolarization. The results suggest that compared with the controls, the "gate" function of the dentate gyrus is compromised in rats with kainate-induced epilepsy, and epileptiform bursts (but not full-length seizure events) can be induced in minislices by repetitive synaptic stimulation at physiological frequencies in the range of hippocampal theta rhythm (i.e., 5-10 Hz).

Both synaptic and intrinsic mechanisms underlie the different properties of population bursts in the hippocampal CA3 area of immature versus adult rats.

Pharmacological blockade of GABA(A) receptors on CA3 pyramidal cells in hippocampal slices from immature rats (i.e. second to third postnatal weeks), compared to CA3 slices from adult rats, is known to cause prolonged burst discharges (i.e. several seconds vs. tens of milliseconds). Synaptic and intrinsic mechanisms responsible for this developmental difference in burst duration were analysed in isolated minislices of the CA3 area. The frequency and amplitude of spontaneous EPSCs in CA3 pyramidal cells were greater in slices from immature than mature rats. In the presence of GABA(A)- and GABA(B)-receptor antagonists, the burst discharges of immature CA3 pyramidal cells were still prolonged in thinner slices (350 microm, vs. 450 microm in adults, to compensate for developmental differences in neuronal density) and in NMDA- and mGlu1-receptor antagonists. The AMPA receptor antagonist DNQX blocked the remaining burst discharges, suggesting that differences in recurrent excitatory circuits contributed to the prolonged bursts of immature CA3 pyramidal cells. In slices from immature versus adult rats, the CA3 recurrent synaptic responses showed potentiation to repetitive stimulation, suggestive of a lower transmitter release probability. The intrinsic firing ability was greater in CA3 pyramidal neurons from immature than adult rats, and the medium-duration afterhyperpolarization was smaller. These data suggest that, compared to adults, the CA3 area of immature rats contains a more robust recurrent excitatory synaptic network, greater intrinsic membrane excitability, and an increased capacity for sustained transmitter release, which together may account for the more prolonged network bursts in immature versus adult CA3.