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1.  Role of GluK1 Kainate Receptors in Seizures, Epileptic Discharges, and Epileptogenesis 
The Journal of Neuroscience  2014;34(17):5765-5775.
Kainate receptors containing the GluK1 subunit have an impact on excitatory and inhibitory neurotransmission in brain regions, such as the amygdala and hippocampus, which are relevant to seizures and epilepsy. Here we used 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a potent and selective agonist of kainate receptors that include the GluK1 subunit, in conjunction with mice deficient in GluK1 and GluK2 kainate receptor subunits to assess the role of GluK1 kainate receptors in provoking seizures and in kindling epileptogenesis. We found that systemic ATPA, acting specifically via GluK1 kainate receptors, causes locomotor arrest and forelimb extension (a unique behavioral characteristic of GluK1 activation) and induces myoclonic behavioral seizures and electrographic seizure discharges in the BLA and hippocampus. In contrast, the proconvulsant activity of systemic AMPA, kainate, and pentylenetetrazol is not mediated by GluK1 kainate receptors, and deletion of these receptors does not elevate the threshold for seizures in the 6 Hz model. ATPA also specifically activates epileptiform discharges in BLA slices in vitro via GluK1 kainate receptors. Olfactory bulb kindling developed similarly in wild-type, GluK1, and GluK2 knock-out mice, demonstrating that GluK1 kainate receptors are not required for epileptogenesis or seizure expression in this model. We conclude that selective activation of kainate receptors containing the GluK1 subunit can trigger seizures, but these receptors are not necessary for seizure generation in models commonly used to identify therapeutic agents for the treatment of epilepsy.
doi:10.1523/JNEUROSCI.5307-13.2014
PMCID: PMC3996208  PMID: 24760837
ATPA; BLA; epilepsy; kainate receptor; kindling; seizure
2.  Perampanel Inhibition of AMPA Receptor Currents in Cultured Hippocampal Neurons 
PLoS ONE  2014;9(9):e108021.
Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM), concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M−1 s−1 and 0.58 s−1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM) was similar at all kainate concentrations (3–100 µM), demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.
doi:10.1371/journal.pone.0108021
PMCID: PMC4168215  PMID: 25229608
3.  Neuroactive Steroids for the Treatment of Status Epilepticus 
Epilepsia  2013;54(0 6):93-98.
SUMMARY
Benzodiazepines are the current first-line standard-of-care treatment for status epilepticus but fail to terminate seizures in about one-third of cases. Synaptic GABAA receptors, which mediate phasic inhibition in central circuits, are the molecular target of benzodiazepines. As status epilepticus progresses, these receptors are internalized and become functionally inactivated, conferring bezodiazepine resistance, which is believed to be a major cause of treatment failure. GABAA receptor positive allosteric modulator neuroactive steroids, such as allopregnanolone, also potentiate synaptic GABAA receptors, but in addition they enhance extrasynaptic GABAA receptors that mediate tonic inhibition. Extrasynaptic GABAA receptors are not internalized and desensitization of these receptors does not occur during continuous seizures in status epilepticus models. Here we review the broad-spectrum antiseizure activity of allopregnanolone in animal seizure models and the evidence for its activity in models of status epilepticus. We also demonstrate that allopregnanolone inhibits ongoing behavioral and electrographic seizures in a model of status epilepticus, even when there is benzodiazepine resistance. Parenteral allopregnanolone may provide an improved treatment for refractory status epilepticus.
doi:10.1111/epi.12289
PMCID: PMC3772544  PMID: 24001085
Refractory status epilepticus; Seizure; Allopregnanolone; Neurosteroid; Allosteric modulator
4.  Neurosteroids as endogenous regulators of seizure susceptibility and use in the treatment of epilepsy 
Epilepsia  2010;51(Suppl 5):84.
SUMMARY
Neurosteroids such as allopregnanolone are positive allosteric modulators of GABAA receptors with powerful antiseizure activity in diverse animal models. Neurosteroids may be endogenous regulators of seizure susceptibility, for example, in catamenial epilepsy. Clinical trials with the synthetic neurosteroid analog ganaxolone in the treatment of refractory partial seizures and infantile spasms have been encouraging. Neurosteroids and analogs such as ganaxolone show promise in the treatment of diverse forms of epilepsy. For an expanded treatment of this topic see Jasper’s basic mechanisms of the epilepsies, 4th ed. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at www.ncbi.nlm.nih.gov/books).
doi:10.1111/j.1528-1167.2010.02870.x
PMCID: PMC4137784  PMID: 25147407
Allopregnanolone; clinical trials; GABAA receptor; Ganaxolone; Infantile spasms; Partial seizures
5.  Epilepsy Therapy Development: Technical and Methodological Issues in Studies with Animal Models 
Epilepsia  2013;54(0 4):13-23.
SUMMARY
The search for new treatments for seizures, epilepsies and their comorbidities faces considerable challenges. Partly, this is due to gaps in our understanding of the etiology and pathophysiology of most forms of epilepsy. An additional challenge is the difficulty to predict the efficacy, tolerability and impact of potential new treatments on epilepsies and comorbidities in humans, using the available resources. Here we provide a summary of the discussions and proposals of the Working Group 2 as presented in the Joint American Epilepsy Society and International League Against Epilepsy Translational Workshop in London (September 2012). We propose methodological and reporting practices that will enhance the uniformity, reliability and reporting of early stage preclinical studies with animal seizure and epilepsy models that aim to develop and evaluate new therapies for seizures or epilepsies, using multi-disciplinary approaches. The topics considered include: (a) implementation of better study design and reporting practices, (b) incorporation in the study design and analysis of covariants that may impact outcomes (including species, age, sex), (c) utilization of approaches to document target relevance, exposure and engagement by the tested treatment, (d) utilization of clinically relevant treatment protocols, (e) optimization of the use of video-EEG recordings to best meet the study goals, and (f) inclusion of outcome measures that address the tolerability of the treatment or study endpoints apart from seizures. We further discuss the different expectations for studies aiming to meet regulatory requirements to obtain approval for clinical testing in humans. Implementation of the rigorous practices discussed in this report will require considerable investment in time, funds and other research resources, which may create challenges for academic researchers seeking to contribute to epilepsy therapy discovery and development. We propose several infrastructure initiatives to overcome these barriers.
doi:10.1111/epi.12295
PMCID: PMC3747731  PMID: 23909850
Pharmacokinetics; video-electroencephalography; tolerability; regulatory requirements; non-pharmacological treatment; infrastructure
6.  Issues related to development of new anti-seizure treatments 
Epilepsia  2013;54(0 4):24-34.
Summary
This report represents a summary of the discussions led by the anti-seizure treatment working group of the ILAE/AES Working Groups joint meeting in London (London Meeting). We review here what is currently known about the pharmacological characteristics of current models of refractory seizures, both for adult and pediatric epilepsy. In addition, we address how the NINDS-funded Anticonvulsant Screening Program (ASP) is evolving to incorporate appropriate animal models in the search for molecules that might be sufficiently novel to warrant further pharmacological development. We also briefly address what we believe is necessary, going forward, to achieve the goal of stopping seizures in all patients, with a call to arms for funding agencies, the pharmaceutical industry, and basic researchers.
doi:10.1111/epi.12296
PMCID: PMC3947404  PMID: 23909851
Anti-seizure drug; pharmacoresistant epilepsy; animal models of epilepsy
7.  Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures 
PLoS ONE  2013;8(12):e80922.
In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.
doi:10.1371/journal.pone.0080922
PMCID: PMC3862847  PMID: 24349022
8.  Tetramethylenedisulfotetramine Alters Ca2+ Dynamics in Cultured Hippocampal Neurons: Mitigation by NMDA Receptor Blockade and GABAA Receptor-Positive Modulation 
Toxicological Sciences  2012;130(2):362-372.
Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca2+ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13–17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca2+ dynamics causing an immediate but transient elevation of neuronal intracellular Ca2+ followed by decreased frequency of Ca2+ oscillations but greater peak amplitude. The effect on Ca2+ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABAA) receptor function. The effect of TETS on Ca2+ dynamics requires activation of N-methyl-d-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca2+ channels. Pretreatment with the GABAA receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca2+ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1µM) and allopregnanolone (0.1µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca2+ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABAA receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.
doi:10.1093/toxsci/kfs244
PMCID: PMC3529644  PMID: 22889812
Ca2+; oscillations; GABAA receptors; microelectrode array; NMDA receptors; rapid throughput assay; tetramethylenedisulfotetramine.
9.  Adjunctive perampanel for refractory partial-onset seizures 
French, Jacqueline A. | Krauss, Gregory L. | Biton, Victor | Squillacote, David | Yang, Haichen | Laurenza, Antonio | Kumar, Dinesh | Rogawski, Michael A. | Campanille, Verónica | Floridia, Jorge | Ilari, Rita | Consalvo, Damián E. | Thomson, Alfredo | Sfaello, Ignacio | Pociecha, Juan | Nieto, Flavia | Firstenfeld, Alfredo | Zuin, Daniel | Mesri, Jacobo | Silva, Walter | Nofal, Pedro | Cristalli, Diana | Clement, Jean-Francois | Hwang, Paul | McLachlan, Richard | Pillay, Neelan | Lasso, Jorge | Peralta, Balduin Lawson | Hernandez, Marcelo Leiva | Tenhamm, Eugenio | Barroso, Noe | Milian, Albino Contreras | Morales, Sarug Reyes | Rodríguez, Ildefonso | Jain, Sanjay | Wilfong, Angus | Kalra, Arun | Renfroe, Ben | Moore, David | Flamini, J. | Klapper, Jack | Szaflarski, Jerzy | Teasley, Jean | Halford, Jonathan | Ferreira, Jose | Goodpasture, Hewitt | Sperling, Michael | Klein, Pavel | Ayala, Ricardo | Brower, Richard | Leroy, Robert | Davis, Ronald | Elterman, Roy | Enlow, Thomas | Sosa, Veronica | Puri, Vinay | Miller, John | Shneker, Bassel | Bautista, Ramon | Chung, Steve | Lesch, David | Steiner, David | Webb, Randall | Armstrong, Robert | Biton, Victor | Krauss, Gregory | Mitchell, Wendy | Hogan, Robert | Knowlton, Robert | Wheless, James | Aung-Din, Ronald | Ali, Imran | Brown, Lawrence | Gruenthal, Michael | Kankirawatana, Pongkiat | Charlet, Michael | Conry, Joan | Owen, Meriem Bensalem | Yerby, Mark | Herrman, Craig | Fessler, Albert | Modur, Pradeep | Miranda, Fernando | King-Stephens, David
Neurology  2012;79(6):589-596.
Objective:
To assess efficacy and safety of once-daily 8 or 12 mg perampanel, a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor antagonist, when added to concomitant antiepileptic drugs (AEDs) in the treatment of drug-resistant partial-onset seizures.
Methods:
This was a multicenter, double-blind, placebo-controlled trial (ClinicalTrials.gov identifier: NCT00699972). Patients (≥12 years, with ongoing seizures despite 1–3 AEDs) were randomized (1:1:1) to once-daily perampanel 8 mg, 12 mg, or placebo. Following baseline (6 weeks), patients entered a 19-week double-blind phase: 6-week titration (2 mg/week increments to target dose) followed by a 13-week maintenance period. Percent change in seizure frequency was the primary endpoint; 50% responder rate was the primary endpoint for EU registration.
Results:
Of 388 patients randomized and treated, 387 provided seizure frequency data. Using this intent-to-treat population over the double-blind phase, the median percent change in seizure frequency was −21.0%, −26.3%, and −34.5% for placebo and perampanel 8 and 12 mg, respectively (p = 0.0261 and p = 0.0158 for 8 and 12 mg vs placebo, respectively). Fifty percent responder rates during the maintenance period were 26.4%, 37.6%, and 36.1%, respectively, for placebo, perampanel 8 mg, and perampanel 12 mg; these differences were not statistically significant for 8 mg (p = 0.0760) or 12 mg (p = 0.0914). Sixty-eight (17.5%) patients discontinued, including 40 (10.3%) for adverse events. Most frequent treatment-emergent adverse events were dizziness, somnolence, irritability, headache, fall, and ataxia.
Conclusions:
This trial demonstrated that once-daily, adjunctive perampanel at doses of 8 or 12 mg improved seizure control in patients with uncontrolled partial-onset seizures. Doses of perampanel 8 and 12 mg were safe, and tolerability was acceptable.
Classification of evidence:
This study provides Class I evidence that once-daily 8 and 12 mg doses of adjunctive perampanel are effective in patients with uncontrolled partial-onset seizures.
doi:10.1212/WNL.0b013e3182635735
PMCID: PMC3413761  PMID: 22843280
10.  Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone 
Human Molecular Genetics  2012;21(13):2923-2935.
Premutation CGG repeat expansions (55–200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca2+ oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-β-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABAA receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.
doi:10.1093/hmg/dds118
PMCID: PMC3373240  PMID: 22466801
11.  11β-Hydoxylase Inhibitors Protect Against Seizures in Mice by Increasing Endogenous Neurosteroid Synthesis 
Neuropharmacology  2011;61(1-2):133-137.
Steroid 11β-hydroxylase (CYP11B1; EC 1.14.15.4) is a mitochondrial enzyme located in the zona fasciculata of the adrenal cortex and also in the brain that mediates the conversion of 11-deoxycortisol to cortisol and 11-deoxycorticosterone (DOC) to corticosterone. Inhibitors of CYP11B1, such as metyrapone and etomidate, reduce glucocorticoid synthesis and raise levels of DOC providing greater availability for metabolic conversion to the GABAA receptor modulating neurosteroid allotetrahydrodeoxycorticosterone (THDOC). Because THDOC is a potent anticonvulsant, it is plausible that CYP11B1 inhibitors could protect against seizures. Here we demonstrate that metyrapone affords dose-dependent protection against 6-Hz seizures 30 min after injection (ED50, 191 mg/kg), but is markedly more potent at 6 h (ED50, 30 mg/kg). Similarly, etomidate is also protective at 30 min and 6 h (ED50 values, 4.5 and 1.7 mg/kg). Finasteride, an inhibitor of neurosteroid synthesis, attenuated the anticonvulsant effects of both CYP11B1 inhibitors at 6 h, but not 30 min following their injection. Plasma THDOC levels measured by liquid chromatography-mass spectrometry were markedly increased 6 h after injection of both CYP11B1 inhibitors and this increase was attenuated by finasteride pretreatment. We conclude that inhibition of CYP11B1 causes delayed seizure protection due to slow build-up of neurosteroids. Early seizure protection is independent of neurosteroids.
doi:10.1016/j.neuropharm.2011.03.019
PMCID: PMC3105122  PMID: 21458468
Neurosteroid; Allotetrahydrodeoxycorticosterone; Steroid 11β-hydroxylase (CYP11B1) inhibitor; Metyrapone; Etomidate; Finasteride
12.  Ganaxolone Suppression of Behavioral and Electrographic Seizures in the Mouse Amygdala Kindling Model 
Epilepsy research  2010;89(2-3):254-260.
Summary
Ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one), a synthetic analog of the endogenous neurosteroid allopregnanolone and a positive allosteric modulator of GABAA receptors, may represent a new treatment approach for epilepsy. Here we demonstrate that pretreatment with ganaxolone (1.25–20 mg/kg, s.c.) causes a dose-dependent suppression of behavioral and electrographic seizures in fully amygdala kindled female mice, with nearly complete seizure protection at the highest dose tested. The ED50 for suppression of behavioral seizures was 6.6 mg/kg. The seizure suppression produced by ganaxolone was comparable to that of clonazepam (ED50, 0.1 mg/kg, s.c.). To the extent that amygdala kindling represents a model of mesial temporal lobe epilepsy, this study supports the utility of ganaxolone in the treatment of patients with temporal lobe seizures.
doi:10.1016/j.eplepsyres.2010.01.009
PMCID: PMC2854307  PMID: 20172694
ganaxolone; neurosteroid; clonazepam; GABAA receptor; amygdala kindling; epilepsy; seizure; female mice
13.  Revisiting AMPA Receptors as an Antiepileptic Drug Target 
Epilepsy Currents  2011;11(2):56-63.
In the 1990s there was intense interest in ionotropic glutamate receptors as therapeutic targets for diverse neurological disorders, including epilepsy. NMDA receptors were thought to play a key role in the generation of seizures, leading to clinical studies of NMDA receptor blocking drugs in epilepsy. Disappointing results dampened enthusiasm for ionotropic glutamate receptors as a therapeutic target. Eventually it became appreciated that another type of ionotropic glutamate receptor, the AMPA receptor, is actually the predominant mediator of excitatory neurotransmission in the central nervous system and moreover that AMPA receptors are critical to the generation and spread of epileptic activity. As drugs became available that selectively target AMPA receptors, it was possible to demonstrate that AMPA receptor antagonists have powerful antiseizure activity in in vitro and in vivo models. A decade later, promising clinical studies with AMPA receptor antagonists, including the potent noncompetitive antagonist perampanel, are once again focusing attention on AMPA receptors as a drug target for epilepsy therapy.
doi:10.5698/1535-7511-11.2.56
PMCID: PMC3117497  PMID: 21686307
16.  Neurosteroids on the Epilepsy Chessboard—Keeping Seizures in Check 
Epilepsy Currents  2010;10(6):161-163.
doi:10.1111/j.1535-7511.2010.01388.x
PMCID: PMC3001027  PMID: 21157546
17.  Pathological alterations in GABAergic interneurons and reduced tonic inhibition in the basolateral amygdala during epileptogenesis 
Neuroscience  2009;163(1):415-429.
An acute brain insult such as traumatic head/brain injury, stroke, or an episode of status epilepticus can trigger epileptogenesis, which, after a latent, seizure-free period, leads to epilepsy. The discovery of effective pharmacological interventions that can prevent the development of epilepsy requires knowledge of the alterations that occur during epileptogenesis in brain regions that play a central role in the induction and expression of epilepsy. In the present study, we investigated pathological alterations in GABAergic interneurons in the rat basolateral amygdala (BLA), and the functional impact of these alterations on inhibitory synaptic transmission, on days 7 to 10 after SE induced by kainic acid. Using design-based stereology combined with GAD67 immunohistochemistry, we found a more extensive loss of GABAergic interneurons compared to the loss of principal cells. Fluoro-Jade C staining showed that neuronal degeneration was still ongoing. These alterations were accompanied by an increase in the levels of glutamate decarboxylase and the α1 subunit of the GABAA receptor, and a reduction in the GluK1 (previously known as GluR5) subunit, as determined by Western blots. Whole-cell recordings from BLA pyramidal neurons showed a significant reduction in the frequency and amplitude of action potential-dependent spontaneous IPSCs, a reduced frequency but not amplitude of miniature IPSCs, and impairment in the modulation of IPSCs via GluK1-containing kainate receptors (GluK1Rs). Thus, in the BLA, GABAergic interneurons are more vulnerable to seizure-induced damage than principal cells. Surviving interneurons increase their expression of glutamate decarboxylase and the α1 GABAA receptor subunit, but this does not compensate for the interneuronal loss; the result is a dramatic reduction of tonic inhibition in the BLA circuitry. As activation of GluK1Rs by ambient levels of glutamate facilitates GABA release, the reduced level and function of these receptors may contribute to the reduction of tonic inhibitory activity. These alterations at a relatively early stage of epileptogenesis may facilitate the progress towards the development of epilepsy.
doi:10.1016/j.neuroscience.2009.06.034
PMCID: PMC2733834  PMID: 19540312
basolateral amygdala; interneurons; epileptogenesis; status epilepticus; kainate receptors; inhibitory synaptic transmission; glutamate decarboxylase; GluR5 subunit; GluK1 subunit; GABAA receptor subunits
18.  Neurosteroid Replacement Therapy for Catamenial Epilepsy 
Summary
Perimenstural catamenial epilepsy, the cyclical occurrence of seizure exacerbations near the time of menstruation, affects a high proportion of women of reproductive age with drug refractory epilepsy. Enhanced seizure susceptibility in perimenstrual catamenial epilepsy is believed to be due to the withdrawal of the progesterone-derived GABAA receptor modulating neurosteroid allopregnanolone as a result of the fall in progesterone at the time of menstruation. Studies in a rat pseudopregnancy model of catamenial epilepsy indicate that following neurosteroid withdrawal there is enhanced susceptibility to chemoconvulsant seizures. There is also a transitory increase in the frequency of spontaneous seizures in epleptic rats that had experienced pilocarpine-induced status epilepticus. In the catamenial epilepsy model, there is a marked reduction in the antiseizure potency of anticonvulsant drugs, including benzodiazepines and valproate, but an increase in the anticonvulsant potency and protective index of neurosteroids such as allopregnanolone and the neurosteroid analog ganaxolone. The enhanced seizure susceptibility and benzodiazepine-resistance following neurosteroid withdrawal may be related to reduced expression and altered kinetics of synaptic GABAA receptors and increased expression of GABAA receptor subunits (such as α4) that confer benzodiazepine insensitivity. The enhanced potency of neurosteroids may be due to a relative increase following neurosteroid withdrawal in the expression of neurosteroid-sensitive δ-subunit-containing perisynaptic/extrasynaptic GABAA receptors. Positive allosteric modulatory neurosteroids and synthetic analogs such as ganaxolone may be administered to prevent catamenial seizure exacerbations, which we refer to as “neurosteroid replacement therapy.”
doi:10.1016/j.nurt.2009.01.006
PMCID: PMC2682439  PMID: 19332335
catamenial epilepsy; progesterone; neurosteroid; allopregnanolone; ganaxolone; GABAA receptor
19.  Convection-Enhanced Delivery in the Treatment of Epilepsy 
Summary
Convection-enhanced delivery (CED) is a novel drug-delivery technique that uses positive hydrostatic pressure to deliver a fluid containing a therapeutic substance by bulk flow directly into the interstitial space within a localized region of the brain parenchyma. CED circumvents the blood-brain barrier and provides a wider, more homogenous distribution than bolus deposition (focal injection) or other diffusion-based delivery approaches. A potential use of CED is for the local delivery of antiseizure agents, which would provide an epilepsy treatment approach that avoids the systemic toxicities of orally administered antiepileptic drugs and bystander effects on non-epileptic brain regions. Recent studies have demonstrated that brief CED infusions of nondiffusible peptides that inhibit the release of excitatory neurotransmitters, including ω-conotoxins and botulinum neurotoxins, can produce long-lasting (weeks to months) seizure protection in the rat amygdala-kindling model. Seizure protection is obtainable without detectable neurological or behavioral side effects. Although conventional diffusible antiepileptic drugs do confer seizure protection when administered locally by CED, the effect is transitory. CED is a potential approach for seizure protection that could represent an alternative to resective surgery in the treatment of focal epilepsies that are resistant to orally-administered antiepileptic drugs. The prolonged duration of action of nondiffusible toxins would allow seizure protection to be maintained chronically with infrequent reinfusions.
doi:10.1016/j.nurt.2009.01.017
PMCID: PMC2753495  PMID: 19332329
Convection-enhanced delivery; drug delivery; ω-conotoxin; botulinum neurotoxin; kindling; antiepileptic drug; epilepsy; seizure
20.  Obituary 
Epilepsy Currents  2009;9(5):141.
doi:10.1111/j.1535-7511.2009.01314.x
PMCID: PMC2759046
21.  New Molecular Targets for Antiepileptic Drugs: α2δ, SV2A, and Kv7/KCNQ/M Potassium Channels 
Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on γ-aminobutyric acid–mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: α2δ, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and Kv7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs.
PMCID: PMC2587091  PMID: 18590620
22.  Intrinsic Severity as a Determinant of Antiepileptic Drug Refractoriness 
Epilepsy Currents  2008;8(5):127-130.
For the most part, resistance to medications in epilepsy is independent of the choice of antiepileptic drug. This simple clinical observation constrains the possible biological mechanisms for drug refractory epilepsy by imposing a requirement to explain resistance for a diverse set of chemical structures that act on an even more varied group of molecular targets. To date, research on antiepileptic drug refractoriness has been guided by the “drug transporter overexpression” and the “reduced drug-target sensitivity” hypotheses. These concepts posit that drug refractoriness is a condition separate from the underlying epilepsy. Inadequacies in both hypotheses mandate a fresh approach to the problem. In this article, we propose a novel approach that considers epilepsy pharmacoresistance in terms of intrinsic disease severity. We suggest that neurobiological factors that confer increased disease severity lead to drug intractability. The occurrence of frequent seizures at disease onset is an important factor that signals increased severity.
doi:10.1111/j.1535-7511.2008.00272.x
PMCID: PMC2566613  PMID: 18852835
23.  Efficacy of the ketogenic diet in the 6-Hz seizure test 
Epilepsia  2007;49(2):334-339.
SUMMARY
Purpose
Since the ketogenic diet is effective in drug-resistant epilepsies, we sought to determine whether it is active in the 6-Hz seizure test, which identifies agents with a broader spectrum of activity than conventional antiepileptic screening tests.
Methods
Male (3–4 week old) NIH Swiss mice were fed a normal or ketogenic diet ad libitum for 2–21 days. The intensity of the corneal stimulation current required to elicit seizures in the 6-Hz test was measured. Blood glucose and β-hydroxybutyrate were measured on the day of seizure testing.
Results
CC50 (current intensity producing seizures in 50% of mice tested) was 50.6 mA and 15 mA in mice fed for 12 days with a ketogenic or normal diet, respectively (p < 0.001). CC50 was elevated in separate experiments after 16, but not 2, 5, and 21 days of ketogenic diet exposure. CC50 values of growing mice fed the normal diet does not differ, indicating CC50 does not vary with mouse weight during a rapid growth phase. β-Hydroxybutyrate was significantly higher, and glucose was significantly lower in mice fed the ketogenic diet than those fed the normal diet. Blood glucose and β-hydroxybutyrate levels did not correlate with CC50.
Discussion
The ketogenic diet significantly elevates the seizure threshold in the 6-Hz test in a time-specific manner. Protection from seizures in this model was not related to level of ketosis. CC50 was insensitive to body weight in mice fed the normal diet, demonstrating that the 6-Hz model can assess anticonvulsant regimens where weight is a confounding factor.
doi:10.1111/j.1528-1167.2007.01430.x
PMCID: PMC2390715  PMID: 18070095
Ketogenic diet; 6-Hz seizure model; Seizure; Intractable epilepsy
24.  Neuroprotective and disease-modifying effects of the ketogenic diet 
Behavioural pharmacology  2006;17(5-6):431-439.
The ketogenic diet has been in clinical use for over 80 years, primarily for the symptomatic treatment of epilepsy. A recent clinical study has raised the possibility that exposure to the ketogenic diet may confer long-lasting therapeutic benefits for patients with epilepsy. Moreover, there is evidence from uncontrolled clinical trials and studies in animal models that the ketogenic diet can provide symptomatic and disease-modifying activity in a broad range of neurodegenerative disorders including Alzheimer’s disease and Parkinson’s disease, and may also be protective in traumatic brain injury and stroke. These observations are supported by studies in animal models and isolated cells that show that ketone bodies, especially β-hydroxybutyrate, confer neuroprotection against diverse types of cellular injury. This review summarizes the experimental, epidemiological and clinical evidence indicating that the ketogenic diet could have beneficial effects in a broad range of brain disorders characterized by the death of neurons. Although the mechanisms are not yet well defined, it is plausible that neuroprotection results from enhanced neuronal energy reserves, which improve the ability of neurons to resist metabolic challenges, and possibly through other actions including antioxidant and anti-inflammatory effects. As the underlying mechanisms become better understood, it will be possible to develop alternative strategies that produce similar or even improved therapeutic effects without the need for exposure to an unpalatable and unhealthy, high-fat diet.
PMCID: PMC2367001  PMID: 16940764
Alzheimer’s disease; cellular energetics; epilepsy; ketone bodies; ketogenic diet; mitochondria; neuroprotection; Parkinson’s disease; stroke; traumatic brain injury
25.  Prolonged Attenuation of Amygdala-Kindled Seizure Measures in Rats by Convection-Enhanced Delivery of the N-Type Calcium Channel Antagonists ω-Conotoxin GVIA and ω-Conotoxin MVIIA 
Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists ω-conotoxin GVIA (ω-CTX-G) and ω-conotoxin MVIIA (ω-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, ω-CTX-G (0.005, 0.05, and 0.5 nmol), ω-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated ω-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of ω-CTX-G and ω-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of ω-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated ω-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.
doi:10.1124/jpet.107.125047
PMCID: PMC2257985  PMID: 17717191

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