Ketogenic diet-mediated seizure reduction preserves CA1 cell numbers in epileptic Kcna1-null mice: An unbiased stereological assessment.

There is growing evidence for the disease-modifying potential of metabolic therapies, including the ketogenic diet (KD), which is used to treat medically intractable epilepsy. However, it remains unclear whether the KD exerts direct effects on histopathological changes in epileptic brain, or whether the changes are a consequence of diet-induced reduction in seizure activity. Here, we used unbiased stereological techniques to quantify the seizure-induced reduction in cell number in the CA1 region of the hippocampus of epileptic Kcna1-null mice and compared the effects of the KD with that of phenobarbital (PB), a widely employed anti-seizure drug. Our data suggest that the anti-seizure activity of the KD or PB was similar. However, CA1 cell numbers of KD-treated hippocampi were not significantly different from those seen in wild-type (WT) mice, whereas CA1 cell counts in standard diet and PB-treated Kcna1-null mice were 23% and 31% lower than WT animals, respectively. These results support the notion that structural protection of cells may involve more than seizure attenuation, and that the KD engages mechanisms that also promote or restore hippocampal morphological integrity.

Progressive cardiorespiratory dysfunction in Kv1.1 knockout mice may provide temporal biomarkers of pending sudden unexpected death in epilepsy (SUDEP): The contribution of orexin.

OBJECTIVE: Immediately preceding sudden unexpected death in epilepsy (SUDEP), patients experienced a final generalized tonic-clonic seizure (GTCS), rapid ventilation, apnea, bradycardia, terminal apnea, and asystole. Whether a progressive pathophysiology develops and increases risk of SUDEP remains unknown. Here, we determined (a) heart rate, respiratory rate, and blood oxygen saturation (SaO2 ) in low-risk and high-risk knockout (KO) mice; and (b) whether blocking receptors for orexin, a cardiorespiratory neuromodulator, influences cardiorespiratory function mice or longevity in high-risk KO mice. METHODS: Heart rate and SaO2 were determined noninvasively with ECGenie and pulse oximetry. Respiration was determined with noninvasive airway mechanics technology. The role of orexin was determined within subject following acute treatment with a dual orexin receptor antagonist (DORA, 100 mg/kg). The number of orexin neurons in the lateral hypothalamus was determined with immunohistochemistry. RESULTS: Intermittent bradycardia was more prevalent in high-risk KO mice, an effect that may be the result of increased parasympathetic drive. High-risk KO mice had more orexin neurons in the lateral hypothalamus. Blocking of orexin receptors differentially influenced heart rate in KO, but not wild-type (WT) mice. When DORA administration increased heart rate, it also decreased heart rate variability, breathing frequency, and/or hypopnea-apnea. Blocking orexin receptors prevented the methacholine (MCh)-induced increase in breathing frequency in KO mice and reduced MCh-induced seizures, via a direct or indirect mechanism. DORA improved oxygen saturation in KO mice with intermittent hypoxia. Daily administration of DORA to high-risk KO mice increased longevity. SIGNIFICANCE: High-risk KO mice have a unique cardiorespiratory phenotype that is characterized by progressive changes in five interdependent endpoints. Blocking of orexin receptors attenuates some of these endpoints and increases longevity, supporting the notion that windows of opportunity for intervention exist in this preclinical SUDEP model.

Adenosine has two faces: Regionally dichotomous adenosine tone in a model of epilepsy with comorbid sleep disorders.

OBJECTIVE: Adenosine participates in maintaining the excitatory/inhibitory balance in neuronal circuits. Studies indicate that adenosine levels in the cortex and hippocampus increase and exert sleep pressure in sleep-deprived and control animals, whereas in epilepsy reduced adenosine tone promotes hyperexcitability. To date, the role of adenosine in pathological conditions that result in both seizures and sleep disorders is unknown. Here, we determined adenosine tone in sleep and seizure regulating brain regions of Kv1.1 knockout (KO) mice, a model of temporal epilepsy with comorbid sleep disorders. METHODS: 1) Reverse phase-high performance liquid chromatography (RP-HPLC) was performed on brain tissue to determine levels of adenosine and adenine nucleotides. 2) Multi-electrode array extracellular electrophysiology was used to determine adenosine tone in the hippocampal CA1 region and the lateral hypothalamus (LH). RESULTS: RP-HPLC indicated a non-significant decrease in adenosine (~50%, p = 0.23) in whole brain homogenates of KO mice. Regional examination of relative levels of adenine nucleotides indicated decreased ATP and increased AMP in the cortex and hippocampus and increased adenosine in cortical tissue. Using electrophysiological and pharmacological techniques, estimated adenosine levels were ~35% lower in the KO hippocampal CA1 region, and 1-2 fold higher in the KO LH. Moreover, the increased adenosine in KO LH contributed to lower spontaneous firing rates of putative wake-promoting orexin/hypocretin neurons. INTERPRETATION: This is the first study to demonstrate a direct correlation of regionally distinct dichotomous adenosine levels in a single model with both epilepsy and comorbid sleep disorders. The weaker inhibitory tone in the dorsal hippocampus is consistent with lower seizure threshold, whereas increased adenosine in the LH is consistent with chronic partial sleep deprivation. This work furthers our understanding of how adenosine may contribute to pathological conditions that underlie sleep disorders within the epileptic brain.

Accumulation of rest deficiency precedes sudden death of epileptic Kv1.1 knockout mice, a model of sudden unexpected death in epilepsy.

OBJECTIVE: Chronic sleep deficiency is associated with early mortality. In the epileptic population, there is a higher prevalence of sleep disorders, and individuals with severe refractory epilepsy are at greater risk of premature mortality than the general population. Sudden unexpected death in epilepsy affects 1:1000 cases of epilepsy each year. Ketogenic diet (KD) treatment is one of the few effective options for refractory seizures. Despite KD reducing seizures and increasing longevity in Kv1.1 knockout (KO) mice, they still succumb to sudden death. This study aims to determine whether (1) the rest profiles of KO and KD-treated KO (KOKD) mice resemble each other as a function of either age or proximity to death and (2) the timing of death correlates with acute or chronic changes in rest. METHODS: Noninvasive actimetry was used to monitor rest throughout the lives of KO and wild-type (WT) littermates administered standard diet or KD. RESULTS: As KO mice age, rest is reduced (P < .0001). Rest is significantly improved in KDKO mice (P < .0001), resembling WT values at several ages. When age is removed as a variable and data are realigned to the day of death, the rest profiles of KO and KOKD groups worsen to similar degrees as a function of proximity to death. The amount of rest acutely is not sensitive to the timing of death, whereas chronic rest deficiency profiles (10-15 days prior to death) of both groups were indistinguishable. Chronic accumulation of rest deficiency over the final 15 days was associated with 75% of deaths. SIGNIFICANCE: Our data suggest that the accumulated rest deficiency is associated with sudden death in Kv1.1 KO mice. These data (1) support the proposed clinical hypothesis that chronic sleep deficiency may be associated with early mortality in epileptic patients and (2) warrant future preclinical and clinical studies on sleep monitoring in epileptic patients.

Respiratory dysfunction progresses with age in Kcna1-null mice, a model of sudden unexpected death in epilepsy.

OBJECTIVE: Increased breathing rate, apnea, and respiratory failure are associated with sudden unexpected death in epilepsy (SUDEP). We recently demonstrated the progressive nature of epilepsy and mortality in Kcna1-/- mice, a model of temporal lobe epilepsy and SUDEP. Here we tested the hypothesis that respiratory dysfunction progresses with age in Kcna1-/- mice, thereby increasing risk of respiratory failure and sudden death (SD). METHODS: Respiratory parameters were determined in conscious mice at baseline and following increasing doses of methacholine (MCh) using noninvasive airway mechanics (NAM) systems. Kcna1+/+ , Kcna1+/- , and Kcna1-/- littermates were assessed during 3 age ranges when up to ~30%, ~55%, and ~90% of Kcna1-/- mice have succumbed to SUDEP: postnatal day (P) 32-36, P40-46, and P48-56, respectively. Saturated arterial O2 (SaO2 ) was determined with pulse oximetry. Lung and brain tissues were isolated and Kcna1 gene and protein expression were evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blot techniques. Airway smooth muscle responsiveness was assessed in isolated trachea exposed to MCh. RESULTS: Kcna1-/- mice experienced an increase in basal respiratory drive, chronic oxygen desaturation, frequent apnea-hypopnea (A-H), an atypical breathing sequence of A-H-tachypnea-A-H, increased tidal volume, and hyperventilation induced by MCh. The MCh-provoked hyperventilation was dramatically attenuated with age. Of interest, only Kcna1-/- mice developed seizures following exposure to MCh. Seizures were provoked by lower concentrations of MCh as Kcna1-/- mice approached SD. MCh-induced seizures experienced by a subset of younger Kcna1-/- mice triggered death. Respiratory parameters of these younger Kcna1-/- mice resembled older near-SD Kcna1-/- mice. Kcna1 gene and protein were not expressed in Kcna1+/+ and Kcna1+/- lungs, and MCh-mediated airway smooth muscle contractions exhibited similar half-maximal effective concentration( EC50 ) in isolated Kcna1+/+ and Kcna1-/- trachea. SIGNIFICANCE: The Kcna1-/- model of SUDEP exhibits progressive respiratory dysfunction, which suggests a potential increased susceptibility for respiratory failure during severe seizures that may result in sudden death.

Synergistic protection against acute flurothyl-induced seizures by adjuvant treatment of the ketogenic diet with the type 2 diabetes drug pioglitazone.

OBJECTIVE: We have previously found that the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) contributes to the mechanism of action of the ketogenic diet (KD), an established treatment for pediatric refractory epilepsy. We have found that the KD increases brain PPARγ and that inhibition or genetic loss of PPARγ prevents the antiseizure effects of the KD on (1) acutely induced seizures in nonepileptic mice and (2) spontaneous recurrent seizures in epileptic mice. Here, we tested the hypothesis that adjuvant treatment of KD-treated mice with a PPARγ agonist, pioglitazone, would result in an additive effect. METHODS: Acute seizures were induced in three groups of C57Bl/6 mice by inhalation exposure to flurothyl gas. In Group 1, mice were weaned onto either a standard diet or KD comprised of a fat:carbohydrate/protein ratio of either 6:1, 3:1, or 1:1 for 2 weeks. In Group 2, vehicle or pioglitazone (0.1, 1, 10, 80 mg/kg) was administered 4 h prior to flurothyl exposure. In Group 3, vehicle or increasing doses of pioglitazone were administered to KD-treated mice 4 h prior to flurothyl exposure. Latency times to clonic seizures and generalized tonic-clonic (GTC) seizures were recorded, and isobolographic analysis was used to determine combinatorial interactions. RESULTS: Neither KD treatment nor pioglitazone alone or in combination affected clonic seizures. However, the latency to GTC seizures was dose-dependently and significantly increased by both KD (~57%, p < 0.05) and pioglitazone (~28%, p < 0.05). Coadministration of an ineffective 1:1 KD and pioglitazone resulted in ~47-55% (p < 0.05) increase in latency to GTC. Isobolographic analysis indicated a synergistic interaction of the KD and pioglitazone. SIGNIFICANCE: These results suggest coadministration may enable reduction of the KD ratio without loss of seizure protection. Such adjuvant treatment could improve quality of life and limit adverse effects of a classic KD or high-dose pioglitazone.

Ketone Bodies as Anti-Seizure Agents.

There is growing evidence that ketone bodies (KB)-derived from fatty acid oxidation and produced during fasting or consumption of high-fat diets-can exert broad neuroprotective effects. With respect to epilepsy, KB (such as ß-hydroxybutyrate or BHB, acetoacetate and acetone) have been shown to block acutely induced and spontaneous recurrent seizures in various animal models. Although the mechanisms underlying the anti-seizure effects of KB have not been fully elucidated, recent experimental studies have invoked ketone-mediated effects on both inhibitory (e.g., GABAergic, purinergic and ATP-sensitive potassium channels) and excitatory (e.g., vesicular glutamate transporters) neurotransmission, as well as mitochondrial targets (e.g., respiratory chain and mitochondrial permeability transition). Moreover, BHB appears to exert both epigenetic (i.e., inhibition of histone deacetylases or HDACs) and anti-inflammatory (i.e., peripheral modulation of hydroxycarboxylic acid receptor and inhibition of the NOD-like receptor protein 3 or NRLP3 inflammasome) activity. While the latter two effects of BHB have yet to be directly linked to ictogenesis and/or epileptogenesis, parallel lines of evidence indicate that HDAC inhibition and a reduction in neuroinflammation alone or collectively can block seizure activity. Nevertheless, the notion that KB are themselves anti-seizure agents requires clinical validation, as prior studies have not revealed a clear correlation between blood ketone levels and seizure control. Notwithstanding this limitation, there is growing evidence that KB are more than just cellular fuels, and can exert profound biochemical, cellular and epigenetic changes favoring an overall attenuation in brain network excitability.

Ketone bodies mediate antiseizure effects through mitochondrial permeability transition.

OBJECTIVE: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. METHODS: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. RESULTS: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. INTERPRETATION: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.

Ketogenic diet treatment increases longevity in Kcna1-null mice, a model of sudden unexpected death in epilepsy.

Individuals with poorly controlled epilepsy have a higher risk for sudden unexpected death in epilepsy (SUDEP). With approximately one third of people with epilepsy not achieving adequate seizure control with current antiseizure drugs, there is a critical need to identify treatments that reduce risk factors for SUDEP. The Kcna1-null mutant mouse models risk factors and terminal events associated with SUDEP. In the current study, we demonstrate the progressive nature of epilepsy and sudden death in this model (mean age of mortality (± SEM), postnatal day [P] 42.8 ± 1.3) and tested the hypothesis that long-term treatment with the ketogenic diet (KD) will prolong the life of Kcna1-null mice. We found that the KD postpones disease progression by delaying the onset of severe seizures and increases the lifespan of these mutant mice by 47%. Future studies are needed to determine the mechanisms underlying the KD effects on longevity.

In vivo ketogenic diet treatment attenuates pathologic sharp waves and high frequency oscillations in in vitro hippocampal slices from epileptic Kv 1.1α knockout mice.

The ketogenic diet (KD) is an effective therapy for pediatric refractory epilepsies; however, whether the KD changes the pathologic network oscillations generated by an epileptic brain remains unknown. We have reported that hippocampal CA3 regions of epileptic Kv1.1α knockout (KO) mice generate pathologic sharp waves (SPWs) and high-frequency oscillations (HFOs) that have higher incidence, longer duration, and fast ripples compared to wild-type (WT). Synaptic activity of hyperexcitable KO mossy fibers significantly decreased CA3 principal cell spike-timing reliability, which contributed to this network pathology. In addition, we have demonstrated that the KD reduces seizures by 75% in KO mice. Here, we determined whether 10- to 14-day in vivo KD treatment exerts disease-modifying effects that alter the spontaneous SPW-HFO complexes generated by the hippocampal CA3 region of KO mice in vitro using extracellular multielectrode array recordings. We found that KD treatment significantly attenuated the pathologic features of KO SPWs and ripples and reduced the incidence of fast ripples. The KD also improved spike-timing reliability of KO CA3 principal cells, decreased mossy fiber excitability, increased mossy fiber-CA3 paired-pulse ratios, and reduced coupling of field excitatory postsynaptic potentials and population spikes in the CA3 region. Collectively, these data indicate that KD treatment modulates CA3-generated pathologic oscillations by dampening hyperactive mossy fiber synapses.

Dietary and Metabolic Approaches for Treating Autism Spectrum Disorders, Affective Disorders and Cognitive Impairment Comorbid with Epilepsy: A Review of Clinical and Preclinical Evidence.

Epilepsy often occurs with other neurological disorders, such as autism, affective disorders, and cognitive impairment. Research indicates that many neurological disorders share a common pathophysiology of dysfunctional energy metabolism, neuroinflammation, oxidative stress, and gut dysbiosis. The past decade has witnessed a growing interest in the use of metabolic therapies for these disorders with or without the context of epilepsy. Over one hundred years ago, the high-fat, low-carbohydrate ketogenic diet (KD) was formulated as a treatment for epilepsy. For those who cannot tolerate the KD, other diets have been developed to provide similar seizure control, presumably through similar mechanisms. These include, but are not limited to, the medium-chain triglyceride diet, low glycemic index diet, and calorie restriction. In addition, dietary supplementation with ketone bodies, polyunsaturated fatty acids, or triheptanoin may also be beneficial. The proposed mechanisms through which these diets and supplements work to reduce neuronal hyperexcitability involve normalization of aberrant energy metabolism, dampening of inflammation, promotion of endogenous antioxidants, and reduction of gut dysbiosis. This raises the possibility that these dietary and metabolic therapies may not only exert anti-seizure effects, but also reduce comorbid disorders in people with epilepsy. Here, we explore this possibility and review the clinical and preclinical evidence where available.

Personalization of SUDEP risk: A survey of transient subclinical comorbid changes.

OBJECTIVE: Preclinical data report within subject modifiable ailments emerge weeks prior to SUDEP, including sleep disorders and cardiorespiratory changes; findings which support anecdotal clinical data. Here, we bridge preclinical findings with future clinical/preclinical studies, and survey whether caretakers or family members of victims noticed transient changes prior to SUDEP. The aim of this pilot study is to identify potential modifiable changes that may synergistically increase SUDEP risk for future research. METHODS: A mobile electronic survey was posted on SUDEP community websites. The survey queried whether changes in seizures, sleep, physical well-being, emotional well-being, cognition, breathing, or heart rate were noticed before SUDEP. RESULTS: The most profound finding was that 85% of victims had multiple transient ailments prior to SUDEP. Changes in seizures (28/54), and sleep (30/58) occurred in more than 50% of the victims and represent the most influential changes identified. The second and third most influential changes were a reduction in physical well-being (25/57) and emotional well-being (26/56). Changes were observed within the last two months of life in approximately one third of the cases, and more than four months prior to SUDEP in approximately one third of cases, indicating a potential time frame for proactive preventative strategies. Respondents also noted changes in cognition (16/55), breathing (9/54) or heart rate (8/55). Data indicate these changes may be associated with increased SUDEP risk within subject. Study limitations include the responses were based on memory, there was a potential for data to be over reported, and caretakers were not prompted to observe changes a priori, thus some existing changes may have gone unnoticed. SIGNIFICANCE: Data support the preclinical findings that transient, subclinical (i.e., not severe enough to require medical intervention), modifiable ailments may increase risk of SUDEP. This suggests that just as an epilepsy type can change over a lifetime and epilepsy type-specific treatments can reduce SUDEP risk, further personalization of SUDEP risk will improve our understanding as to whether variables contribute to risk differently across lifespan. Thus, with a dynamic capacity to change, differing factors may contribute to the distribution of risk probability within an individual at any given time. Understanding whether different combinations of transient changes are specific to epilepsy type, age, or sex needs to be determined to move the field forward in hopes of developing a personalized approach to preventative strategies.

Loss of the Kv1.1 potassium channel promotes pathologic sharp waves and high frequency oscillations in in vitro hippocampal slices.

In human disease, channelopathies involving functional reduction of the delayed rectifier potassium channel α-subunit Kv1.1 - either by mutation or autoimmune inhibition - result in temporal lobe epilepsy. Kv1.1 is prominently expressed in the axons of the hippocampal tri-synaptic pathway, suggesting its absence will result in widespread effects on normal network oscillatory activity. Here, we performed in vitro extracellular recordings using a multielectrode array to determine the effects of loss of Kv1.1 on spontaneous sharp waves (SPWs) and high frequency oscillations (HFOs). We found that Kcna1-null hippocampi generate SPWs and ripples (80-200Hz bandwidth) with a 50% increased rate of incidence and 50% longer duration, and that epilepsy-associated pathologic HFOs in the fast ripple bandwidth (200-600Hz) are also present. Furthermore, Kcna1-null CA3 has enhanced coupling of excitatory inputs and population spike generation and CA3 principal cells have reduced spike timing reliability. Removing the influence of mossy fiber and perforant path inputs by micro-dissecting the Kcna1-null CA3 region mostly rescued the oscillatory behavior and improved spike timing. We found that Kcna1-null mossy fibers and medial perforant path axons are hyperexcitable and produce greater pre- and post-synaptic responses with reduced paired-pulse ratios suggesting increased neurotransmitter release at these terminals. These findings were recapitulated in wild-type slices exposed to the Kv1.1 inhibitor dendrotoxin-κ. Collectively, these data indicate that loss of Kv1.1 enhances synaptic release in the CA3 region, which reduces spike timing precision of individual neurons leading to disorganization of network oscillatory activity and promotes the emergence of fast ripples.

Orexin receptors regulate hippocampal sharp wave-ripple complexes in ex vivo slices.

Orexin is a neuromodulatory peptide produced by lateral hypothalamic orexin neurons and binds to G-protein-coupled orexin-1 receptor and orexin-2 receptors. Whether orexin modulates learning and memory is not fully understood. Orexin has biphasic effects on learning and memory: promoting learning and memory at homeostatic levels and inhibiting at supra- and sub-homeostatic levels. Hippocampal sharp wave-ripples encode memory information and are essential for memory consolidation and retrieval. The role of orexin on sharp wave-ripples in hippocampal CA1 remains unknown. Here, we used multi-electrode array recordings in acute ex vivo hippocampal slices to determine the effects of orexin receptor antagonists on sharp wave-ripples. Bath-application of either the orexin-1 receptor antagonist N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea (SB-334867) or the orexin-2 receptor antagonist N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA) reduced sharp wave and ripple incidence, sharp wave amplitude, and sharp wave duration. SB-334867 and EMPA effects on sharp wave amplitude and duration were equivalent, whereas EMPA exhibited a greater reduction of sharp wave and ripple incidence. EMPA also increased ripple duration, whereas SB-334867 had no effect. Inhibition of both orexin receptors with a dual orexin receptor antagonist N-[1,1'-Biphenyl]-2-yl-1-[2-[(1-methyl-1H-benzimidazol-2-yl)thio]acetyl-2-pyrrolidinedicarboxamide (TCS-1102) had effects similar to EMPA, however, sharp wave amplitude and duration were unaffected. Region-specific expression of orexin receptors suggests orexin may regulate sharp wave generation in CA3, dentate gyrus-mediated sharp wave modification, sharp wave propagation to CA1, and local ripple emergence in CA1. Our study indicates an orexin contribution to hippocampal sharp wave-ripple complexes and suggests a mechanism by which sub-homeostatic concentrations of orexin may inhibit learning and memory function.

Ascorbic Acid Reduces Neurotransmission, Synaptic Plasticity, and Spontaneous Hippocampal Rhythms in In Vitro Slices.

Ascorbic acid (AA; a.k.a. vitamin C) is well known for its cellular protection in environments of high oxidative stress. Even though physiological concentrations of AA in the brain are significant (0.2-10 mM), surprisingly little is known concerning the role of AA in synaptic neurotransmission under normal, non-disease state conditions. Here, we examined AA effects on neurotransmission, plasticity and spontaneous network activity (i.e., sharp waves and high frequency oscillations; SPW-HFOs), at the synapse between area 3 and 1 of the hippocampal cornu ammonis region (CA3 and CA1) using an extracellular multi-electrode array in in vitro mouse hippocampal slices. We found that AA decreased evoked field potentials (fEPSPs, IC50 = 0.64 mM) without affecting V50s or paired pulse facilitation indicating normal neurotransmitter release mechanisms. AA decreased presynaptic fiber volleys but did not change fiber volley-to-fEPSP coupling, suggesting reduced fEPSPs resulted from decreased fiber volleys. Inhibitory effects were also observed in CA1 stratum pyramidale where greater fEPSPs were required for population spikes in the presence of AA suggesting an impact on the intrinsic excitability of neurons. Other forms of synaptic plasticity and correlates of memory (i.e., short- and long-term potentiation) were also significantly reduced by AA as was the incidence of spontaneous SPW-HFOs. AA decreased SPW amplitude with a similar IC50 as fEPSPs (0.65 mM). Overall, these results indicate that under normal conditions AA significantly regulates neurotransmission, plasticity, and network activity by limiting excitability. Thus, AA may participate in refinement of signal processing and memory formation, as well as protecting against pathologic excitability.

Functional MRI Correlates of Carbon Dioxide Chemosensing in Persons With Epilepsy.

Objectives: Sudden unexpected death in epilepsy (SUDEP) is a catastrophic epilepsy outcome for which there are no reliable premortem imaging biomarkers of risk. Percival respiratory depression is seen in monitored SUDEP and near SUDEP cases, and abnormal chemosensing of raised blood carbon dioxide (CO2) is thought to contribute. Damage to brainstem respiratory control and chemosensing structures has been demonstrated in structural imaging and neuropathological studies of SUDEP. We hypothesized that functional MRI (fMRI) correlates of abnormal chemosensing are detectable in brainstems of persons with epilepsy (PWE) and are different from healthy controls (HC). Methods: We analyzed fMRI BOLD activation and brain connectivity in 10 PWE and 10 age- and sex-matched HCs during precisely metered iso-oxic, hypercapnic breathing challenges. Segmented brainstem responses were of particular interest, along with characterization of functional connectivity metrics between these structures. Regional BOLD activations during hypercapnic challenges were convolved with hemodynamic responses, and the resulting activation maps were passed on to group-level analyses. For the functional connectivity analysis, significant clusters from BOLD results were used as seeds. Each individual seed time-series activation map was extracted for bivariate correlation coefficient analyses to study changes in brain connectivity between PWE and HCs. Results: (1) Greater brainstem BOLD activations in PWE were observed compared to HC during hypercapnic challenges in several structures with respiratory/chemosensing properties. Group comparison between PWE vs. HC showed significantly greater activation in the dorsal raphe among PWE (p < 0.05) compared to HCs. (2) PWE had significantly greater seed-seed connectivity and recruited more structures during hypercapnia compared to HC. Significance: The results of this study show that BOLD responses to hypercapnia in human brainstem are detectable and different in PWE compared to HC. Increased dorsal raphe BOLD activation in PWE and increased seed-seed connectivity between brainstem and adjacent subcortical areas may indicate abnormal chemosensing in these individuals. Imaging investigation of brainstem respiratory centers involved in respiratory regulation in PWE is an important step toward identifying suspected dysfunction of brainstem breathing control that culminates in SUDEP and deserve further study as potential imaging SUDEP biomarkers.

L-Type calcium channel blockade reduces network activity in human epileptic hypothalamic hamartoma tissue.

PURPOSE: Human hypothalamic hamartomas (HHs) are associated with gelastic seizures, intrinsically epileptogenic, and notoriously refractory to medical therapy. We previously reported that the L-type calcium channel antagonist nifedipine blocks spontaneous firing and γ-aminobutyric acid (GABA)(A)-induced depolarization of single cells in HH tissue slices. In this study, we examined whether blocking L-type calcium channels attenuates emergent activity of HH neuronal networks. METHODS: A high-density multielectrode array was used to record extracellular signals from surgically resected HH tissue slices. High-frequency oscillations (HFOs, ripples and fast ripples), field potentials, and multiunit activity (MUA) were studied (1) under normal and provoked [4-aminopyridine (4-AP)] conditions; and (2) following nifedipine treatment. KEY FINDINGS: Spontaneous activity occurred during normal artificial cerebrospinal fluid (aCSF) conditions. Nifedipine reduced the total number and duration of HFOs, abolished the association of HFOs with field potentials, and increased the inter-HFO burst intervals. Notably, the number of active regions was decreased by 45 ± 9% (mean ± SEM) after nifedipine treatment. When considering electrodes that detected activity, nifedipine increased MUA in 58% of electrodes and reduced the number of field potentials in 67% of electrodes. Provocation with 4-AP increased the number of events and, as the number of electrodes that detected activity increased 248 ± 62%, promoted tissue-wide propagation of activity. During provocation with 4-AP, nifedipine effectively reduced HFOs, the association of HFOs with field potentials, field potentials, MUA, and the number of active regions, and limited propagation. SIGNIFICANCE: This is the first study to report (1) the presence of HFOs in human subcortical epileptic brain tissue in vitro; (2) the modulation of "pathologic" high-frequency oscillations (i.e., fast ripples) in human epileptic tissue by L-type calcium channel blockers; and (3) the modulation of network physiology and synchrony of emergent activity in human epileptic tissue following blockade of L-type calcium channels. Attenuation of activity in HH tissue during normal and provoked conditions supports a potential therapeutic usefulness of L-type calcium channel blockers in epileptic patients with HH.

Carbamazepine Reduces Sharp Wave-Ripple Complexes and Exerts Synapse-Specific Inhibition of Neurotransmission in Ex Vivo Hippocampal Slices.

Higher therapeutic concentrations of the antiseizure medication carbamazepine (CBZ) are associated with cognitive side effects. Hippocampal sharp wave-ripple complexes (SPW-Rs) are proposed to participate in memory consolidation during periods of quiet and slow-wave sleep. SPW-Rs are generated in the CA3 region and are regulated by multiple synaptic inputs. Here, we used a multi-electrode array to determine the effects of CBZ on SPW-Rs and synaptic transmission at multiple hippocampal synapses. Our results demonstrate that CBZ reduced SPW-Rs at therapeutically relevant concentrations (IC50 = 37 µM) and altered the core characteristics of ripples, important for information processing and consolidation. Moreover, CBZ inhibited neurotransmission in a synapse-specific manner. CBZ inhibition was most potent at the medial-perforant-path-to-CA3 and mossy-fiber-to-CA3 synapses (IC50s ~ 30 and 60 µM, respectively) and least potent at medial-perforant-path-to-dentate granule cell synapses (IC50 ~ 120 µM). These results suggest that the synapse-specific CBZ inhibition of neurotransmission reduces SPW-Rs and that the CBZ inhibition of SPW-Rs may underlie the cognitive impairments observed with therapeutic doses of CBZ.

Pharmacoresponsiveness of spontaneous recurrent seizures and the comorbid sleep disorder of epileptic Kcna1-null mice.

Drug resistant epilepsy affects ∼30% of people with epilepsy and is associated with epilepsy syndromes with frequent and multiple types of seizures, lesions or cytoarchitectural abnormalities, increased risk of mortality and comorbidities such as cognitive impairment and sleep disorders. A limitation of current preclinical models is that spontaneous seizures with comorbidities take time to induce and test, thus making them low-throughput. Kcna1-null mice exhibit all the characteristics of drug resistant epilepsy with spontaneous seizures and comorbidities occurring naturally; thus, we aimed to determine whether they also demonstrate pharmacoresistanct seizures and the impact of medications on their sleep disorder comorbidity. In this exploratory study, Kcna1-null mice were treated with one of four conventional antiseizure medications, carbamazepine, levetiracetam, phenytoin, and phenobarbital using a moderate throughput protocol (vehicle for 2 days followed by 2 days of treatment with high therapeutic doses selected based on published data in the 6 Hz model of pharmacoresistant seizures). Spontaneous recurrent seizures and vigilance states were recorded with video-EEG/EMG. Carbamazepine, levetiracetam and phenytoin had partial efficacy (67%, 75% and 33% were seizure free, respectively), whereas phenobarbital was fully efficacious and conferred seizure freedom to all mice. Thus, seizures of Kcna1-null mice appear to be resistant to three of the drugs tested. Levetiracetam failed to affect sleep architecture, carbamazepine and phenytoin had moderate effects, and phenobarbital, as predicted, restored sleep architecture. Data suggest Kcna1-null mice may be a moderate throughput model of drug resistant epilepsy useful in determining mechanisms of pharmacoresistance and testing novel therapeutic strategies.

Proceedings of the Sleep and Epilepsy Workshop: Section 3 Mortality: Sleep, Night, and SUDEP.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy. Likely pathophysiological mechanisms include seizure-induced cardiac and respiratory dysregulation. A frequently identified feature in SUDEP cases is that they occur at night. This raises the question of a role for sleep state in regulating of SUDEP. An association with sleep has been identified in a number of studies with patients and in animal models. The focus of this section of the Sleep and Epilepsy Workshop was on identifying and understanding the role for sleep and time of day in the pathophysiology of SUDEP.