Adenosine A1 receptor-mediated protection of mouse hippocampal synaptic transmission against oxygen and/or glucose deprivation: a comparative study.

Adenosine receptors are widely expressed in the brain, and adenosine is a key bioactive substance for neuroprotection. In this article, we clarify systematically the role of adenosine A1 receptors during a range of timescales and conditions when a significant amount of adenosine is released. Using acute hippocampal slices obtained from mice that were wild type or null mutant for the adenosine A1 receptor, we quantified and characterized the impact of varying durations of experimental ischemia, hypoxia, and hypoglycemia on synaptic transmission in the CA1 subregion. In normal tissue, these three stressors rapidly and markedly reduced synaptic transmission, and only treatment of sufficient duration led to incomplete recovery. In contrast, inactivation of adenosine A1 receptors delayed and/or lessened the reduction in synaptic transmission during all three stressors and reduced the magnitude of the recovery significantly. We reproduced the responses to hypoxia and hypoglycemia by applying an adenosine A1 receptor antagonist, validating the clear effects of genetic receptor inactivation on synaptic transmission. We found activation of adenosine A1 receptor inhibited hippocampal synaptic transmission during the acute phase of ischemia, hypoxia, or hypoglycemia and caused the recovery from synaptic impairment after these three stressors using genetic mutant. These studies quantify the neuroprotective role of the adenosine A1 receptor during a variety of metabolic stresses within the same recording system.NEW & NOTEWORTHY Deprivation of oxygen and/or glucose causes a rapid adenosine A1 receptor-mediated decrease in synaptic transmission in mouse hippocampus. We quantified adenosine A1 receptor-mediated inhibition during and synaptic recovery after ischemia, hypoxia, and hypoglycemia of varying durations using a genetic mutant and confirmed these findings using pharmacology. Overall, using the same recording conditions, we found the acute response and the neuroprotective ability of the adenosine A1 receptor depended on the type and duration of deprivation event.

Ketogenic diet sensitizes glucose control of hippocampal excitability.

A high-fat low-carbohydrate ketogenic diet (KD) is an effective treatment for refractory epilepsy, yet myriad metabolic effects in vivo have not been reconciled clearly with neuronal effects. A KD limits blood glucose and produces ketone bodies from ß-oxidation of lipids. Studies have explored changes in ketone bodies and/or glucose in the effects of the KD, and glucose is increasingly implicated in neurological conditions. To examine the interaction between altered glucose and the neural effects of a KD, we fed rats and mice a KD and restricted glucose in vitro while examining the seizure-prone CA3 region of acute hippocampal slices. Slices from KD-fed animals were sensitive to small physiological changes in glucose, and showed reduced excitability and seizure propensity. Similar to clinical observations, reduced excitability depended on maintaining reduced glucose. Enhanced glucose sensitivity and reduced excitability were absent in slices obtained from KD-fed mice lacking adenosine A1 receptors (A1Rs); in slices from normal animals effects of the KD could be reversed with blockers of pannexin-1 channels, A1Rs, or KATP channels. Overall, these studies reveal that a KD sensitizes glucose-based regulation of excitability via purinergic mechanisms in the hippocampus and thus link key metabolic and direct neural effects of the KD.

The impact of methodology on the reproducibility and rigor of DNA methylation data.

Epigenetic modifications are crucial for normal development and implicated in disease pathogenesis. While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Here we show that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. We examined genome-wide DNA methylation and gene expression profiles of hippocampal tissues from wild-type rats housed in three independent laboratories using nearly identical conditions. Reduced-representation bisulfite sequencing and RNA-seq respectively identified 3852 differentially methylated and 1075 differentially expressed genes between laboratories, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. Our study demonstrates that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. This is particularly meaningful for neurological studies in animal models, in which baseline parameters between experimental groups are difficult to control. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results.

Metabolic autocrine regulation of neurons involves cooperation among pannexin hemichannels, adenosine receptors, and KATP channels.

Metabolic perturbations that decrease or limit blood glucose-such as fasting or adhering to a ketogenic diet-reduce epileptic seizures significantly. To date, the critical links between altered metabolism and decreased neuronal activity remain unknown. More generally, metabolic changes accompany numerous CNS disorders, and the purines ATP and its core molecule adenosine are poised to translate cell energy into altered neuronal activity. Here we show that nonpathological changes in metabolism induce a purinergic autoregulation of hippocampal CA3 pyramidal neuron excitability. During conditions of sufficient intracellular ATP, reducing extracellular glucose induces pannexin-1 hemichannel-mediated ATP release directly from CA3 neurons. This extracellular ATP is dephosphorylated to adenosine, activates neuronal adenosine A(1) receptors, and, unexpectedly, hyperpolarizes neuronal membrane potential via ATP-sensitive K(+) channels. Together, these data delineate an autocrine regulation of neuronal excitability via ATP and adenosine in a seizure-prone subregion of the hippocampus and offer new mechanistic insight into the relationship between decreased glucose and increased seizure threshold. By establishing neuronal ATP release via pannexin hemichannels, and hippocampal adenosine A(1) receptors coupled to ATP-sensitive K(+) channels, we reveal detailed information regarding the relationship between metabolism and neuronal activity and new strategies for adenosine-based therapies in the CNS.

A ketogenic diet reduces long-term potentiation in the dentate gyrus of freely behaving rats.

Ketogenic diets are very low in carbohydrates and can reduce epileptic seizures significantly. This dietary therapy is particularly effective in pediatric and drug-resistant epilepsy. Hypothesized anticonvulsant mechanisms of ketogenic diets focus on increased inhibition and/or decreased excitability/excitation. Either of these consequences might not only reduce seizures, but also could affect normal brain function and synaptic plasticity. Here, we characterized effects of a ketogenic diet on hippocampal long-term potentiation, a widely studied form of synaptic plasticity. Adult male rats were placed on a control or ketogenic diet for 3 wk before recording. To maintain the most physiological conditions possible, we assessed synaptic transmission and plasticity using chronic in vivo recordings in freely behaving animals. Rats underwent stereotaxic surgery to chronically implant a recording electrode in the hippocampal dentate gyrus and a stimulating electrode in the perforant path; they recovered for 1 wk. After habituation and stable baseline recording, 5-Hz theta-burst stimulation was delivered to induce long-term potentiation. All animals showed successful plasticity, demonstrating that potentiation was not blocked by the ketogenic diet. Compared with rats fed a control diet, rats fed a ketogenic diet demonstrated significantly diminished long-term potentiation. This decreased potentiation lasted for at least 48 h. Reduced potentiation in ketogenic diet-fed rats is consistent with a general increase in neuronal inhibition (or decrease in excitability) and decreased seizure susceptibility. A better understanding of the effects of ketogenic diets on synaptic plasticity and learning is important, as diet-based therapy is often prescribed to children with epilepsy.

Ketogenic diet effects on inflammatory allodynia and ongoing pain in rodents.

Ketogenic diets are very low carbohydrate, high fat, moderate protein diets used to treat medication-resistant epilepsy. Growing evidence suggests that one of the ketogenic diet's main mechanisms of action is reducing inflammation. Here, we examined the diet's effects on experimental inflammatory pain in rodent models. Young adult rats and mice were placed on the ketogenic diet or maintained on control diet. After 3-4 weeks on their respective diets, complete Freund's adjuvant (CFA) was injected in one hindpaw to induce inflammation; the contralateral paw was used as the control. Tactile sensitivity (von Frey) and indicators of spontaneous pain were quantified before and after CFA injection. Ketogenic diet treatment significantly reduced tactile allodynia in both rats and mice, though with a species-specific time course. There was a strong trend to reduced spontaneous pain in rats but not mice. These data suggest that ketogenic diets or other ketogenic treatments might be useful treatments for conditions involving inflammatory pain.

Differential ketogenic diet-induced shift in CSF lipid/carbohydrate metabolome of pediatric epilepsy patients with optimal vs. no anticonvulsant response: a pilot study.

BACKGROUND: The low carbohydrate, high fat ketogenic diet can be an effective anticonvulsant treatment in some pediatric patients with pharmacoresistant epilepsy. Its mechanism(s) of action, however, remain uncertain. Direct sampling of cerebrospinal fluid before and during metabolic therapy may reveal key changes associated with differential clinical outcomes. We characterized the relationship between seizure responsiveness and changes in lipid and carbohydrate metabolites. METHODS: We performed metabolomic analysis of cerebrospinal fluid samples taken before and during ketogenic diet treatment in patients with optimal response (100% seizure remission) and patients with no response (no seizure improvement) to search for differential diet effects in hallmark metabolic compounds in these two groups. Optimal responders and non-responders were similar in age range and included males and females. Seizure types and the etiologies or syndromes of epilepsy varied but did not appear to differ systematically between responders and non-responders. RESULTS: Analysis showed a strong effect of ketogenic diet treatment on the cerebrospinal fluid metabolome. Longitudinal and between-subjects analyses revealed that many lipids and carbohydrates were changed significantly by ketogenic diet, with changes typically being of larger magnitude in responders. Notably, responders had more robust changes in glucose and the ketone bodies ß-hydroxybutyrate and acetoacetate than non-responders; conversely, non-responders had significant increases in fructose and sorbose, which did not occur in responders. CONCLUSIONS: The data suggest that a differential and stronger metabolic response to the ketogenic diet may predict a better anticonvulsant response, and such variability is likely due to inherent biological factors of individual patients. Strategies to boost the metabolic response may be beneficial.

Wdfy3 regulates glycophagy, mitophagy, and synaptic plasticity.

Autophagy is essential to cell function, as it enables the recycling of intracellular constituents during starvation and in addition functions as a quality control mechanism by eliminating spent organelles and proteins that could cause cellular damage if not properly removed. Recently, we reported on Wdfy3's role in mitophagy, a clinically relevant macroautophagic scaffold protein that is linked to intellectual disability, neurodevelopmental delay, and autism spectrum disorder. In this study, we confirm our previous report that Wdfy3 haploinsufficiency in mice results in decreased mitophagy with accumulation of mitochondria with altered morphology, but expanding on that observation, we also note decreased mitochondrial localization at synaptic terminals and decreased synaptic density, which may contribute to altered synaptic plasticity. These changes are accompanied by defective elimination of glycogen particles and a shift to increased glycogen synthesis over glycogenolysis and glycophagy. This imbalance leads to an age-dependent higher incidence of brain glycogen deposits with cerebellar hypoplasia. Our results support and further extend Wdfy3's role in modulating both brain bioenergetics and synaptic plasticity by including glycogen as a target of macroautophagic degradation.

Adenosine and Ketogenic Treatments.

It is well known that the neuromodulator adenosine, acting through the adenosine A1 receptor subtype, can limit or stop seizures. In 2008, adenosine was proposed as a key component of the anticonvulsant mechanism of the ketogenic diet (KD), a very low carbohydrate diet that can be highly effective in drug-refractory epilepsy. In this study, we review the accumulated data on the intersection among adenosine, ketosis, and anticonvulsant/antiepileptogenic effects. In several rodent models of epilepsy and seizures, antiseizure effects of ketogenic treatments (the KD itself, exogenous ketone bodies, medium-chain triglycerides or fatty acids) are reversed by administration of an adenosine A1 receptor antagonist. In addition, KD treatment elevates extracellular adenosine and tissue adenosine content in brain. Efforts to maintain or mimic a ketogenic milieu in brain slices reveal a state of reduced excitability produced by pre- and postsynaptic adenosine A1 receptor-based effects. Long-lasting seizure reduction may be due to adenosine-based epigenetic effects. In conclusion, there is accumulating evidence for an adenosinergic anticonvulsant action in the ketogenic state. In some cases, the main trigger is mildly but consistently lowered glucose in the brain. More research is needed to investigate the importance of adenosine in the antiepileptogenic and neuroprotective effects of these treatments. Future research may begin to investigate alternative adenosine-promoting strategies to enhance the KD or to find use as treatments themselves.

A unifying mechanism of ketogenic diet action: The multiple roles of nicotinamide adenine dinucleotide.

The ability of a ketogenic diet to treat seizures and render a neuronal network more resistant to strong electrical activity has been observed for a century in clinics and for decades in research laboratories. Alongside ongoing efforts to understand how this therapy works to stop seizures, metabolic health is increasingly appreciated as critical buffer to resisting and recovering from acute and chronic disease. Accordingly, links between metabolism and health, and the broader emerging impact of the ketogenic diet in improving diverse metabolic, immunological and neurological conditions, have served to intensify the search for its key and/or common mechanisms. Here we review diverse evidence for increased levels of NAD+, and thus an altered ratio of NAD+/NADH, during metabolic therapy with a ketogenic diet. We propose this as a potential unifying mechanism, and highlight some of the evidence linking altered NAD+/NADH with reduced seizures and with a range of short and long-term changes associated with the beneficial effects of a ketogenic diet. An increase in NAD+/NADH is consistent with multiple lines of evidence and hypotheses, and therefore we suggest that increased NAD+ may be a common mechanism underlying beneficial effects of ketogenic diet therapy.

Reduced-volume cues effectively support fear conditioning despite sleep deprivation.

Sleep deprivation impairs contextual but not cued learned fear, and it has been suggested that this pattern reflects an insensitivity of the amygdala to sleep loss. The lack of effect of sleep deprivation on cued conditioning, however, might simply be due to the strong attention drawn by the typically loud cue tone. We reduced tone volume from our standard 80 dB to either 70 or 60 dB, to test if reduced cue volume allowed effects of sleep deprivation to be detected. Using the platform-over-water method, male C57BL/6 mice were sleep-deprived for 24 h; control mice were moved to novel cages for 24 h. Mice then underwent fear conditioning with a standard "delay" protocol, and were tested for contextual and cued learning the next day. A control group received no footshock during conditioning. In the cue test, and for both cue volumes, SD had no effect on freezing to the tone, which was very robust in conditioned mice regardless of sleep treatment. As expected, freezing to the tone in the no-shock groups was essentially absent. Also, freezing prior to the tone was low in all mice. At the lowest volume, the tone was only ~10 dB above background noise. 24 h sleep deprivation, however, blocked contextual fear in the same mice. These results support a pattern of sleep deprivation sparing amygdaloid function but impairing hippocampal function.

A ketogenic diet diminishes behavioral responses to cocaine in young adult male and female rats.

Ketogenic diets (KDs) are high fat, low carbohydrate formulations traditionally used to treat epilepsy; more recently, KDs have shown promise for a wide range of other neurological disorders. Drug addiction studies suggest that repeated exposure to drugs of abuse, including cocaine, results in a suite of neurobiological changes that includes neuroinflammation, decreased glucose metabolism, and disordered neurotransmission. Given that KDs positively regulate these factors, we addressed whether administration of a KD has potential as a novel therapy for drug addiction. In this study, male and female Sprague-Dawley rats were placed on a KD or a control diet (CD), beginning at five weeks of age and continuing through the end of behavioral testing. Three weeks after initiation of dietary treatments, rats received daily i.p. injections of cocaine (15 mg/kg) or saline vehicle for one week, were drug free for a subsequent week, and then all animals received a final challenge injection of 15 mg/kg cocaine. In the absence of cocaine injections, stereotyped locomotor responses were minimal and were unaffected by dietary treatment. In contrast, both males and females fed a KD exhibited decreased cocaine-induced stereotyped responses as compared to CD-fed rats. The sensitization of ambulatory responses was also disrupted in KD-fed rats. These results suggest that KDs directly impact dopamine-mediated behaviors, and hence may hold potential as a therapy for drug addiction.

Aspects of learned fear related to the hippocampus are sleep-dependent.

Reduced sleep interferes with contextual but not cued learned fear, and it was suggested that this selectivity reflects underlying neural substrates. The apparent lack of contextual fear in sleep-deprived animals, however, could be secondary to hyperactivity. Also, changing the parameters of cued conditioning can change the neural pathways involved, such that some types of cued fear might be sensitive to sleep loss. To address these issues, we measured fear expressed with conditioned defecation as well as behavior, and used a trace cued learning paradigm. Using the platform-over-water method, male Sprague-Dawley rats were continuously sleep-deprived for 3 days, or for 20 h/day for 3 days. Animals then underwent fear conditioning, and were tested for learning the next day. Sleep-deprived or -restricted animals showed a lack of contextual fear at testing, as conditioned freezing and defecation were minimal. Sleep deprivation also blocked cued fear after trace conditioning. Therefore, reduced sleep impairs contextual learning, and impairs cued learning only when the hippocampus is involved. The data support a model in which sleep loss interferes with hippocampal function while sparing amygdala function.

Ketogenic Diet Modulates NAD+-Dependent Enzymes and Reduces DNA Damage in Hippocampus.

The ketogenic diet's (KD) anti-seizure effects have long been documented. Recently, its therapeutic potential in multiple neurodegenerative and neurodevelopmental disorders has emerged. Yet experimental evidence for a fundamental mechanism underlying beneficial effects across numerous diseases remains lacking. We previously showed that feeding rats a KD produced an early (within 2 days) and persistent elevation of hippocampal nicotinamide adenine dinucleotide+ (NAD+), an essential metabolic coenzyme and signaling molecule. NAD+ is a marker of cellular health and a substrate for enzymes implicated in longevity and DNA damage repair such as sirtuins and poly-ADP ribose polymerase-1 (PARP-1). As a result, activation of NAD+-dependent enzymes' downstream pathways could be the origin of KD's broad beneficial effects. Here rats were fed ad libitum regular chow or KD for 2 days or 3 weeks and the levels of hippocampal sirtuins, PARP-1, and the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine were quantified. We found a significant immediate and persistent increase in the collective activity of nuclear sirtuin enzymes, and a significant augmentation of Sirt1 mRNA at 2 days. Levels of PARP-1 and 8-hydroxy-2'-deoxyguanosine decreased after 2 days of treatment and further declined at 3 weeks. Our data show that a KD can rapidly modulate energy metabolism by acting on NAD+-dependent enzymes and their downstream pathways. Thus, therapy with a KD can potentially enhance brain health and increase overall healthspan via NAD+-related mechanisms that render cells more resilient against DNA damage and a host of metabolic, epileptic, neurodegenerative, or neurodevelopmental insults.

Menthol and nicotine oppositely modulate body temperature in the rat.

Menthol is a prominent additive in many tobacco products. To investigate possible interactions with nicotine, (-)-menthol (200 or 400 mg/kg) and (-)-nicotine (0.5 mg/kg) were injected subcutaneously in rats, and body temperature, which is modulated by brain nicotinic acetylcholine receptors, was measured. Nicotine caused robust (-1.6 degrees C) hypothermia, the magnitude and time course of which was not altered by menthol pretreatment. Menthol alone produced mild (0.4-0.8 degrees C) hyperthermia, which was not secondary to locomotor activation. Nicotine and menthol influence body temperature independently and oppositely; menthol does not appear to influence the function of the central nicotinic receptors that control body temperature.

Ketogenic diet improves behaviors in a maternal immune activation model of autism spectrum disorder.

Prenatal factors influence autism spectrum disorder (ASD) incidence in children and can increase ASD symptoms in offspring of animal models. These may include maternal immune activation (MIA) due to viral or bacterial infection during the first trimesters. Unfortunately, regardless of ASD etiology, existing drugs are poorly effective against core symptoms. For nearly a century a ketogenic diet (KD) has been used to treat seizures, and recent insights into mechanisms of ASD and a growing recognition that immune/inflammatory conditions exacerbate ASD risk has increased interest in KD as a treatment for ASD. Here we studied the effects of KD on core ASD symptoms in offspring exposed to MIA. To produce MIA, pregnant C57Bl/6 mice were injected with the viral mimic polyinosinic-polycytidylic acid; after weaning offspring were fed KD or control diet for three weeks. Consistent with an ASD phenotype of a higher incidence in males, control diet-fed MIA male offspring were not social and exhibited high levels of repetitive self-directed behaviors; female offspring were unaffected. However, KD feeding partially or completely reversed all MIA-induced behavioral abnormalities in males; it had no effect on behavior in females. KD-induced metabolic changes of reduced blood glucose and elevated blood ketones were quantified in offspring of both sexes. Prior work from our laboratory and others demonstrate KDs improve relevant behaviors in several ASD models, and here we demonstrate clear benefits of KD in the MIA model of ASD. Together these studies suggest a broad utility for metabolic therapy in improving core ASD symptoms, and support further research to develop and apply ketogenic and/or metabolic strategies in patients with ASD.

Ketone-Based Metabolic Therapy: Is Increased NAD+ a Primary Mechanism?

The ketogenic diet's (KD) anticonvulsant effects have been well-documented for nearly a century, including in randomized controlled trials. Some patients become seizure-free and some remain so after diet cessation. Many recent studies have explored its expanded therapeutic potential in diverse neurological disorders, yet no mechanism(s) of action have been established. The diet's high fat, low carbohydrate composition reduces glucose utilization and promotes the production of ketone bodies. Ketone bodies are a more efficient energy source than glucose and improve mitochondrial function and biogenesis. Cellular energy production depends on the metabolic coenzyme nicotinamide adenine dinucleotide (NAD), a marker for mitochondrial and cellular health. Furthermore, NAD activates downstream signaling pathways (such as the sirtuin enzymes) associated with major benefits such as longevity and reduced inflammation; thus, increasing NAD is a coveted therapeutic endpoint. Based on differential NAD+ utilization during glucose- vs. ketone body-based acetyl-CoA generation for entry into the tricarboxylic cycle, we propose that a KD will increase the NAD+/NADH ratio. When rats were fed ad libitum KD, significant increases in hippocampal NAD+/NADH ratio and blood ketone bodies were detected already at 2 days and remained elevated at 3 weeks, indicating an early and persistent metabolic shift. Based on diverse published literature and these initial data we suggest that increased NAD during ketolytic metabolism may be a primary mechanism behind the beneficial effects of this metabolic therapy in a variety of brain disorders and in promoting health and longevity.

Ketogenic diets improve behaviors associated with autism spectrum disorder in a sex-specific manner in the EL mouse.

The core symptoms of autism spectrum disorder are poorly treated with current medications. Symptoms of autism spectrum disorder are frequently comorbid with a diagnosis of epilepsy and vice versa. Medically-supervised ketogenic diets are remarkably effective nonpharmacological treatments for epilepsy, even in drug-refractory cases. There is accumulating evidence that supports the efficacy of ketogenic diets in treating the core symptoms of autism spectrum disorders in animal models as well as limited reports of benefits in patients. This study tests the behavioral effects of ketogenic diet feeding in the EL mouse, a model with behavioral characteristics of autism spectrum disorder and comorbid epilepsy. Male and female EL mice were fed control diet or one of two ketogenic diet formulas ad libitum starting at 5weeks of age. Beginning at 8weeks of age, diet protocols continued and performance of each group on tests of sociability and repetitive behavior was assessed. A ketogenic diet improved behavioral characteristics of autism spectrum disorder in a sex- and test-specific manner; ketogenic diet never worsened relevant behaviors. Ketogenic diet feeding improved multiple measures of sociability and reduced repetitive behavior in female mice, with limited effects in males. Additional experiments in female mice showed that a less strict, more clinically-relevant diet formula was equally effective in improving sociability and reducing repetitive behavior. Taken together these results add to the growing number of studies suggesting that ketogenic and related diets may provide significant relief from the core symptoms of autism spectrum disorder, and suggest that in some cases there may be increased efficacy in females.

Eliminating the adrenal stress response does not affect sleep deprivation-induced acquisition deficits in the water maze.

Sleep deprivation impairs spatial learning in the rat. Sleep deprivation, however, also causes stress and stress itself can interfere with spatial learning. To address this confound, sleep deprivation effects on Morris water maze training were studied in intact rats and in rats in which the adrenal stress response had been eliminated by adrenalectomy. Stable, physiological levels of corticosterone were maintained in adrenalectomized rats with an implanted pellet. Training occurred 6-7 days after surgery. Seventy-two hours sleep deprivation by the platform-over-water method just prior to training slowed, but did not block, learning. In particular, the robust savings between trials 1 and 2 of the first set found in home cage rats was not present in sleep-deprived rats. Adrenalectomy/corticosterone replacement surgery did not modify the effect of sleep deprivation on acquisition rate, demonstrating that the deficits in spatial task acquisition due to pre-training sleep deprivation are not secondary to the adrenal stress response.

Metabolic Therapy for Temporal Lobe Epilepsy in a Dish: Investigating Mechanisms of Ketogenic Diet using Electrophysiological Recordings in Hippocampal Slices.

The hippocampus is prone to epileptic seizures and is a key brain region and experimental platform for investigating mechanisms associated with the abnormal neuronal excitability that characterizes a seizure. Accordingly, the hippocampal slice is a common in vitro model to study treatments that may prevent or reduce seizure activity. The ketogenic diet is a metabolic therapy used to treat epilepsy in adults and children for nearly 100 years; it can reduce or eliminate even severe or refractory seizures. New insights into its underlying mechanisms have been revealed by diverse types of electrophysiological recordings in hippocampal slices. Here we review these reports and their relevant mechanistic findings. We acknowledge that a major difficulty in using hippocampal slices is the inability to reproduce precisely the in vivo condition of ketogenic diet feeding in any in vitro preparation, and progress has been made in this in vivo/in vitro transition. Thus far at least three different approaches are reported to reproduce relevant diet effects in the hippocampal slices: (1) direct application of ketone bodies; (2) mimicking the ketogenic diet condition during a whole-cell patch-clamp technique; and (3) reduced glucose incubation of hippocampal slices from ketogenic diet-fed animals. Significant results have been found with each of these methods and provide options for further study into short- and long-term mechanisms including Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, vesicular glutamate transporter (VGLUT), pannexin channels and adenosine receptors underlying ketogenic diet and other forms of metabolic therapy.