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The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force established the TASK3 working groups to create common data elements (CDEs) for various preclinical epilepsy research disciplines. This is the second in a two-part series of omics papers, with the other including genomics, transcriptomics, and epigenomics. The aim of the CDEs was to improve the standardization of experimental designs across a range of epilepsy research-related methods. We have generated CDE tables with key parameters and case report forms (CRFs) containing the essential contents of the study protocols for proteomics, lipidomics, and metabolomics of samples from rodent models and people with epilepsy. We discuss the important elements that need to be considered for the proteomics, lipidomics, and metabolomics methodologies, providing a rationale for the parameters that should be documented., (© 2022 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force established the TASK3 working groups to create common data elements (CDEs) for various preclinical epilepsy research disciplines. The aim of the CDEs is to improve the standardization of experimental designs across a range of epilepsy research-related methods. Here, we have generated CDE tables with key parameters and case report forms (CRFs) containing the essential contents of the study protocols for genomics, transcriptomics, and epigenomics in rodent models of epilepsy, with a specific focus on adult rats and mice. We discuss the important elements that need to be considered for genomics, transcriptomics, and epigenomics methodologies, providing a rationale for the parameters that should be collected. This is the first in a two-part series of omics papers with the second installment to cover proteomics, lipidomics, and metabolomics in adult rodents., (© 2022 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

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., (© 2022. The Author(s).)

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 β-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.

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.

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., (Copyright © 2020. Published by Elsevier B.V.)

It is well known that the neuromodulator adenosine, acting through the adenosine A 1 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 A 1 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 A 1 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., Competing Interests: No competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

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 A 1 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 A 1 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 A 1 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 A 1 receptor antagonist, validating the clear effects of genetic receptor inactivation on synaptic transmission. We found activation of adenosine A 1 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 A 1 receptor during a variety of metabolic stresses within the same recording system. NEW & NOTEWORTHY Deprivation of oxygen and/or glucose causes a rapid adenosine A 1 receptor-mediated decrease in synaptic transmission in mouse hippocampus. We quantified adenosine A 1 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 A 1 receptor depended on the type and duration of deprivation event.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Epilepsy is a common neurologic disorder in humans and domesticated canines. In both species the etiology is diverse and complex, and even with medication a significant portion of the population does not experience sufficient seizure control and/or has unacceptable side effects. Humans often try alternatives such as dietary therapy or brain surgery, but in dogs, brain surgery is rarely an option and, despite potential benefits, there are no standard recommendations for a dietary approach. Herein we describe 2 retrospective case studies detailing the effects of homemade diets prepared for dogs with uncontrolled epileptic seizures and/or toxic side effects of medication. Basic recipes are provided for each formula-a high-fat "ketogenic" diet and a partial "whole food" diet. Carbohydrate content was reduced or controlled, and in one case this was proven to be essential for seizure control: ingesting carbohydrates would reverse the benefits of the diet and precipitate a seizure. Both dogs experienced fewer seizures and side effects when eating these modified diets compared to when they were administered antiepileptic drugs, including complete cessation of seizures for extended periods. Practical advantages and success of these homemade dietary interventions highlight the potential for diet-based metabolic therapy as a treatment option for seizures not only in humans but also in dogs., Competing Interests: 7Author Beth Zupec‐Kania is the sole proprietor of Ketogenic Therapies, LLC. The remaining authors have no conflicts of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Metabolic dysfunction can underlie seizure disorders, and metabolism-based treatments can afford seizure control and promote homeostasis. This relationship between metabolism and the risk of sporadic seizures was observed historically with the clinical success of a low-carbohydrate, high-fat, ketosis-inducing ketogenic diet - a treatment that remains relevant today, and one that has been shown to be effective against medically refractory epilepsy. Mechanisms underlying the success of the ketogenic diet are a topic of intense research efforts - not only because of proven success in arresting treatment-resistant seizures, but also because recent evidence suggests that altering metabolism with a ketogenic diet enables a homeostatic state in the brain that is less excitable, and hence raises the threshold for seizure genesis. Metabolic therapy with a ketogenic diet has been shown to normalize a range of abnormal physiological and behavioral parameters and may also make the central nervous system more resilient to other insults or physiological stresses. Because the therapeutic ability of such a diet may be more limited than a drug because of a dose "ceiling", investigations are underway to develop and test analogous or supplemental approaches. In addition, significant efforts have been made to demonstrate broader applications of metabolic therapy in promoting health and preventing disease, including conditions where epileptic seizures manifest in a comorbid fashion., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A subset of neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating depth and frequency of breathing in response to changes in tissue CO 2 /H + . The activity of chemosensitive RTN neurons is also subject to modulation by CO 2 /H + -dependent purinergic signaling. However, mechanisms contributing to purinergic regulation of RTN chemoreceptors are not entirely clear. Recent evidence suggests adenosine inhibits RTN chemoreception in vivo by activation of A1 receptors. The goal of this study was to characterize effects of adenosine on chemosensitive RTN neurons and identify intrinsic and synaptic mechanisms underlying this response. Cell-attached recordings from RTN chemoreceptors in slices from rat or wild-type mouse pups (mixed sex) show that exposure to adenosine (1 µM) inhibits chemoreceptor activity by an A1 receptor-dependent mechanism. However, exposure to a selective A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX; 30 nM) alone did not potentiate CO 2 /H + -stimulated activity, suggesting activation of A1 receptors does not limit chemoreceptor activity under these reduced conditions. Whole-cell voltage-clamp from chemosensitive RTN neurons shows that exposure to adenosine activated an inward rectifying K + conductance, and at the network level, adenosine preferentially decreased frequency of EPSCs but not IPSCs. These results show that adenosine activation of A1 receptors inhibits chemosensitive RTN neurons by direct activation of a G-protein-regulated inward-rectifier K + (GIRK)-like conductance, and presynaptically, by suppression of excitatory synaptic input to chemoreceptors.

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.

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.

Autism spectrum disorder (ASD) is characterized by deficits in sociability and communication, and increased repetitive and/or restrictive behaviors. While the etio-pathogenesis of ASD is unknown, clinical manifestations are diverse and many possible genetic and environmental factors have been implicated. As such, it has been a great challenge to identify key neurobiological mechanisms and to develop effective treatments. Current therapies focus on co-morbid conditions (such as epileptic seizures and sleep disturbances) and there is no cure for the core symptoms. Recent studies have increasingly implicated mitochondrial dysfunction in ASD. The fact that mitochondria are an integral part of diverse cellular functions and are susceptible to many insults could explain how a wide range of factors can contribute to a consistent behavioral phenotype in ASD. Meanwhile, the high-fat, low-carbohydrate ketogenic diet (KD), used for nearly a century to treat medically intractable epilepsy, has been shown to enhance mitochondrial function through a multiplicity of mechanisms and affect additional molecular targets that may address symptoms and comorbidities of ASD. Here, we review the evidence for the use of metabolism-based therapies such as the KD in the treatment of ASD as well as emerging co-morbid models of epilepsy and autism. Future research directions aimed at validating such therapeutic approaches and identifying additional and novel mechanistic targets are also discussed.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (K ATP ) channels, vesicular glutamate transporter (VGLUT), pannexin channels and adenosine receptors underlying ketogenic diet and other forms of metabolic therapy.

Epilepsy is a highly prevalent seizure disorder which tends to progress in severity and become refractory to treatment. Yet no therapy is proven to halt disease progression or to prevent the development of epilepsy. Because a high fat low carbohydrate ketogenic diet (KD) augments adenosine signaling in the brain and because adenosine not only suppresses seizures but also affects epileptogenesis, we hypothesized that a ketogenic diet might prevent epileptogenesis through similar mechanisms. Here, we tested this hypothesis in two independent rodent models of epileptogenesis. Using a pentylenetetrazole kindling paradigm in mice, we first show that a KD, but not a conventional antiepileptic drug (valproic acid), suppressed kindling-epileptogenesis. Importantly, after treatment reversal, increased seizure thresholds were maintained in those animals kindled in the presence of a KD, but not in those kindled in the presence of valproic acid. Next, we tested whether a KD can halt disease progression in a clinically relevant model of progressive epilepsy. Epileptic rats that developed spontaneous recurrent seizures after a pilocarpine-induced status epilepticus were treated with a KD or control diet (CD). Whereas seizures progressed in severity and frequency in the CD-fed animals, KD-fed animals showed a prolonged reduction of seizures, which persisted after diet reversal. KD-treatment was associated with increased adenosine and decreased DNA methylation, the latter being maintained after diet discontinuation. Our findings demonstrate that a KD prevented disease progression in two mechanistically different models of epilepsy, and suggest an epigenetic mechanism underlying the therapeutic effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ketogenic diets are low-carbohydrate, sufficient protein, high-fat diets with anticonvulsant activity used primarily as a treatment for pediatric epilepsy. The anticonvulsant mechanism is thought to involve elevating inhibition and/or otherwise limiting excitability in the brain. Such a mechanism, however, might also significantly affect normal brain activity and limit synaptic plasticity, effects that would be important to consider in the developing brain. To assess ketogenic diet effects on synaptic transmission and plasticity, electrophysiological recordings were performed at the perforant path/dentate gyrus synapse in awake, freely-behaving juvenile male rats. Electrodes were implanted 1 week prior to recording. Animals were fed regular chow or a ketogenic diet ad libitum for 3 weeks before recording. Although the ketogenic diet did not significantly alter baseline excitability (assessed by input-output curves) or short-term plasticity (using the paired-pulse ratio), it did reduce the magnitude of long-term potentiation at all poststimulation timepoints out to the last time measured (48 h). The results suggest an effect of ketogenic diet-feeding on the induction magnitude but not the maintenance of long-term potentiation. The lack of effect of the diet on baseline transmission and the paired-pulse ratio suggests a mechanism that limits excitation preferentially in conditions of strong stimulation, consonant with clinical reports in which the ketogenic diet alleviates seizures without a major impact on normal brain activity. Limiting plasticity in a seizure-susceptible network may limit seizure-induced epileptogenesis which may subserve the ongoing benefit of the ketogenic diet in epilepsy., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

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 β-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., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as "inchworming," was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

Adenosine receptors are a powerful therapeutic target for regulating epileptic seizures. As a homeostatic bioenergetic network regulator, adenosine is perfectly suited to establish or restore an ongoing balance between excitation and inhibition, and its anticonvulsant efficacy is well established. There is evidence for the involvement of multiple adenosine receptor subtypes in epilepsy, but in particular the adenosine A1 receptor subtype can powerfully and bidirectionally regulate seizure activity. Mechanisms that regulate adenosine itself are increasingly appreciated as targets to thus influence receptor activity and seizure propensity. Taken together, established evidence for the powerful potential of adenosine-based epilepsy therapies and new strategies to influence receptor activity can combine to capitalize on this endogenous homeostatic neuromodulator., (© 2014 Elsevier Inc. All rights reserved.)

Ketogenic diets are well established as a successful anticonvulsant therapy. Based on overlap between mechanisms postulated to underlie pain and inflammation, and mechanisms postulated to underlie therapeutic effects of ketogenic diets, recent studies have explored the ability for ketogenic diets to reduce pain. Here we review clinical and basic research thus far exploring the impact of a ketogenic diet on thermal pain, inflammation, and neuropathic pain.

Neuronal excitability of the brain and ongoing homeostasis depend not only on intrinsic neuronal properties, but also on external environmental factors; together these determine the functionality of neuronal networks. Homeostatic factors become critically important during epileptogenesis, a process that involves complex disruption of self-regulatory mechanisms. Here we focus on the bioenergetic homeostatic network regulator adenosine, a purine nucleoside whose availability is largely regulated by astrocytes. Endogenous adenosine modulates complex network function through multiple mechanisms including adenosine receptor-mediated pathways, mitochondrial bioenergetics, and adenosine receptor-independent changes to the epigenome. Accumulating evidence from our laboratories shows that disruption of adenosine homeostasis plays a major role in epileptogenesis. Conversely, we have found that reconstruction of adenosine's homeostatic functions provides new hope for the prevention of epileptogenesis. We will discuss how adenosine-based therapeutic approaches may interfere with epileptogenesis on an epigenetic level, and how dietary interventions can be used to restore network homeostasis in the brain. We conclude that reconstruction of homeostatic functions in the brain offers a new conceptual advance for the treatment of neurological conditions which goes far beyond current target-centric treatment approaches.

Autism spectrum disorders share three core symptoms: impaired sociability, repetitive behaviors and communication deficits. Incidence is rising, and current treatments are inadequate. Seizures are a common comorbidity, and since the 1920's a high-fat, low-carbohydrate ketogenic diet has been used to treat epilepsy. Evidence suggests the ketogenic diet and analogous metabolic approaches may benefit diverse neurological disorders. Here we show that a ketogenic diet improves autistic behaviors in the BTBR mouse. Juvenile BTBR mice were fed standard or ketogenic diet for three weeks and tested for sociability, self-directed repetitive behavior, and communication. In separate experiments, spontaneous intrahippocampal EEGs and tests of seizure susceptibility (6 Hz corneal stimulation, flurothyl, SKF83822, pentylenetetrazole) were compared between BTBR and control (C57Bl/6) mice. Ketogenic diet-fed BTBR mice showed increased sociability in a three-chamber test, decreased self-directed repetitive behavior, and improved social communication of a food preference. Although seizures are a common comorbidity with autism, BTBR mice fed a standard diet exhibit neither spontaneous seizures nor abnormal EEG, and have increased seizure susceptibility in just one of four tests. Thus, behavioral improvements are dissociable from any antiseizure effect. Our results suggest that a ketogenic diet improves multiple autistic behaviors in the BTBR mouse model. Therefore, ketogenic diets or analogous metabolic strategies may offer novel opportunities to improve core behavioral symptoms of autism spectrum disorders.

Unlabelled: Ketogenic diets (KDs) are high-fat, low-carbohydrate formulations effective in treating medically refractory epilepsy, and recently we demonstrated lowered sensitivity to thermal pain in rats fed a KD for 3 to 4 weeks. Regarding anticonvulsant and hypoalgesic mechanisms, theories are divided as to direct effects of increased ketones and/or decreased glucose, metabolic hallmarks of these diets. To address this point, we characterized the time course of KD-induced thermal hypoalgesia, ketosis, and lowered glucose in young male rats fed ad libitum on normal chow or KDs. A strict 6.6:1 (fat:[carbohydrates + protein], by weight) KD increased blood ketones and reduced blood glucose by 2 days of feeding, but thermal hypoalgesia did not appear until 10 days. Thus, ketosis and decreased glucose are not sufficient for hypoalgesia. After feeding a 6.6:1 KD for 19 days, decreased thermal pain sensitivity and changes in blood chemistry reversed 1 day after return to normal chow. Effects were consistent between 2 different diet formulations: a more moderate and clinically relevant KD formula (3.0:1) produced hypoalgesia and similar changes in blood chemistry as the 6.6:1 diet, thus increasing translational potential. Furthermore, feeding the 3.0:1 diet throughout an extended protocol (10-11 weeks) revealed that significant hypoalgesia and increased ketones persisted whereas low glucose did not, demonstrating that KD-induced hypoalgesia does not depend on reduced glucose. In separate experiments we determined that effects on thermal pain responses were not secondary to motor or cognitive changes. Together, these findings dissociate diet-related changes in nociception from direct actions of elevated ketones or decreased glucose, and suggest mechanisms with a slower onset in this paradigm. Overall, our data indicate that metabolic approaches can relieve pain., Perspective: Chronic pain is a common and debilitating condition. We show that a KD, a high-fat, very low carbohydrate diet well known for treating epilepsy, lowers sensitivity to thermal pain in rats. This reduced pain is not temporally correlated with hallmark diet-induced changes in blood glucose and ketones., (Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.)

In rodents, insufficient adenosine produces behavioral and physiological symptoms consistent with several comorbidities of autism. In rodents and humans, stimuli postulated to increase adenosine can ameliorate these comorbidities. Because adenosine is a broad homeostatic regulator of cell function and nervous system activity, increasing adenosine's influence might be a new therapeutic target for autism with multiple beneficial effects. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

HIV-1-associated neurocognitive disorder (HAND) is a syndrome that ranges clinically from subtle neuropsychological impairments to profoundly disabling HIV-associated dementia. Not only is the pathogenesis of HAND unclear, but also effective treatments are unavailable. The HIV-1 transactivator of transcription protein (HIV-1 Tat) is strongly implicated in the pathogenesis of HAND, in part, because of its well-characterized ability to directly excite neurons and cause neurotoxicity. Consistent with previous findings from others, we demonstrate here that HIV-1 Tat induced neurotoxicity, increased intracellular calcium, and disrupted a variety of mitochondria functions, such as reducing mitochondrial membrane potential, increasing levels of reactive oxygen species, and decreasing bioenergetic efficiency. Of therapeutic importance, we show that treatment of cultured neurons with ketone bodies normalized HIV-1 Tat induced changes in levels of intracellular calcium, mitochondrial function, and neuronal cell death. Ketone bodies are normally produced in the body and serve as alternative energy substrates in tissues including brain and can cross the blood-brain barrier. Ketogenic strategies have been used clinically for treatment of neurological disorders and our current results suggest that similar strategies may also provide clinical benefits in the treatment of HAND., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A(1) receptor (A(1)R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A(1)Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A(1)R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A(1)Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A link between metabolism and brain function is clear. Since ancient times, epileptic seizures were noted as treatable with fasting, and historical observations of the therapeutic benefits of fasting on epilepsy were confirmed nearly 100 years ago. Shortly thereafter a high fat, low-carbohydrate ketogenic diet (KD) debuted as a therapy to reduce seizures. This strict regimen could mimic the metabolic effects of fasting while allowing adequate caloric intake for ongoing energy demands. Today, KD therapy, which forces predominantly ketone-based rather than glucose-based metabolism, is now well-established as highly successful in reducing seizures. Cellular metabolic dysfunction in the nervous system has been recognized as existing side-by-side with nervous system disorders - although often with much less obvious cause-and-effect as the relationship between fasting and seizures. Rekindled interest in metabolic and dietary therapies for brain disorders complements new insight into their mechanisms and broader implications. Here we describe the emerging relationship between a KD and adenosine as a way to reset brain metabolism and neuronal activity and disrupt a cycle of dysfunction. We also provide an overview of the effects of a KD on cognition and recent data on the effects of a KD on pain, and explore the relative time course quantified among hallmark metabolic changes, altered neuron function and altered animal behavior assessed after diet administration. We predict continued applications of metabolic therapies in treating dysfunction including and beyond the nervous system.

The neuromodulator adenosine is an endogenous sleep promoter, neuroprotector and anticonvulsant, and people with autism often suffer from sleep disruption and/or seizures. We hypothesized that increasing adenosine can decrease behavioral symptoms of autism spectrum disorders, and, based on published research, specific physiological stimuli are expected to increase brain adenosine. To test the relationship between adenosine and autism, we developed a customized parent-based questionnaire to assess child participation in activities expected to influence adenosine and quantify behavioral changes following these experiences. Parents were naive to study hypotheses and all conditions were pre-assigned. Results demonstrate significantly better behavior associated with events pre-established as predicted to increase rather than decrease or have no influence on adenosine. Understanding the physiological relationship between adenosine and autism could open new therapeutic strategies--potentially preventing seizures, improving sleep, and reducing social and behavioral dysfunction., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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 diets are high in fat and low in carbohydrates, and have long been used as an anticonvulsant therapy for drug-intractable and pediatric epilepsy. Additionally, ketogenic diets have been shown to provide neuroprotective effects against acute and chronic brain injury, including beneficial effects in various rodent models of neurodegeneration. Huntington's disease is a progressive neurodegenerative disease characterized by neurological, behavioral and metabolic dysfunction, and ketogenic diets have been shown to increase energy molecules and mitochondrial function. We tested the effects of a ketogenic diet in a transgenic mouse model of Huntington's disease (R6/2 1J), with a focus on life-long behavioral and physiological effects. Matched male and female wild-type and transgenic mice were maintained on a control diet or were switched to a ketogenic diet fed ad libitum starting at six weeks of age. We found no negative effects of the ketogenic diet on any behavioral parameter tested (locomotor activity and coordination, working memory) and no significant change in lifespan. Progressive weight loss is a hallmark feature of Huntington's disease, yet we found that the ketogenic diet-which generally causes weight loss in normal animals-delayed the reduction in body weight of the transgenic mice. These results suggest that metabolic therapies could offer important benefits for Huntington's disease without negative behavioral or physiological consequences., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.

INTRODUCTION: Despite epilepsy being one of the most prevalent neurological disorders, one third of all patients with epilepsy cannot adequately be treated with available antiepileptic drugs. One of the significant causes for the failure of conventional pharmacotherapeutic treatment is the development of pharmacoresistance in many forms of epilepsy. The problem of pharmacoresistance has called for the development of new conceptual strategies that improve future drug development efforts. AREAS COVERED: A thorough review of the recent literature on pharmacoresistance in epilepsy was completed and select examples were chosen to highlight the mechanisms of pharmacoresistance in epilepsy and to demonstrate how those mechanistic findings might lead to improved treatment of pharmacoresistant epilepsy. The reader will gain a thorough understanding of pharmacoresistance in epilepsy and an appreciation of the limitations of conventional drug development strategies. EXPERT OPINION: Conventional drug development efforts aim to achieve specificity of symptom control by enhancing the selectivity of drugs acting on specific downstream targets; this conceptual strategy bears the undue risk of development of pharmacoresistance. Modulation of homeostatic bioenergetic network regulation is a novel conceptual strategy to affect whole neuronal networks synergistically by mobilizing multiple endogenous biochemical and receptor-dependent molecular pathways. In our expert opinion we conclude that homeostatic bioenergetic network regulation might thus be used as an innovative strategy for the control of pharmacoresistant seizures. Recent focal adenosine augmentation strategies support the feasibility of this strategy.

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.

Marijuana is a widely used drug that impairs memory through interaction between its psychoactive constituent, Delta-9-tetrahydrocannabinol (Delta(9)-THC), and CB(1) receptors (CB1Rs) in the hippocampus. CB1Rs are located on Schaffer collateral (Sc) axon terminals in the hippocampus, where they inhibit glutamate release onto CA1 pyramidal neurons. This action is shared by adenosine A(1) receptors (A1Rs), which are also located on Sc terminals. Furthermore, A1Rs are tonically activated by endogenous adenosine (eADO), leading to suppressed glutamate release under basal conditions. Colocalization of A1Rs and CB1Rs, and their coupling to shared components of signal transduction, suggest that these receptors may interact. We examined the roles of A1Rs and eADO in regulating CB1R inhibition of glutamatergic synaptic transmission in the rodent hippocampus. We found that A1R activation by basal or experimentally increased levels of eADO reduced or eliminated CB1R inhibition of glutamate release, and that blockade of A1Rs with caffeine or other antagonists reversed this effect. The CB1R-A1R interaction was observed with the agonists WIN55,212-2 and Delta(9)-THC and during endocannabinoid-mediated depolarization-induced suppression of excitation. A1R control of CB1Rs was stronger in the C57BL/6J mouse hippocampus, in which eADO levels were higher than in Sprague Dawley rats, and the eADO modulation of CB1R effects was absent in A1R knock-out mice. Since eADO levels and A1R activation are regulated by homeostatic, metabolic, and pathological factors, these data identify a mechanism in which CB1R function can be controlled by the brain adenosine system. Additionally, our data imply that caffeine may potentiate the effects of marijuana on hippocampal function.

Ketogenic diets are high in fat and low in carbohydrates and represent a well-established and effective treatment alternative to anti-epileptic drugs. Ketogenic diets are used for the management of a variety of difficult-to-treat or intractable seizure disorders, especially pediatric refractory epilepsy. However, it has been shown that this dietary therapy can reduce seizures in people of all ages, and ketogenic diets are being applied to other prevalent medical conditions such as diabetes. Although used effectively to treat epilepsy for nearly 90 years, the mechanism(s) by which ketogenic diets work to reduce seizures remain ill-understood. One mechanism receiving increased attention is based on findings that ketogenic diets increase the brain energy molecule ATP, and may also increase the levels and actions of the related endogenous inhibitory neuromodulator adenosine. ATP and adenosine have both been identified as important modulators of seizures; seizures increase the actions of these purines, these purines regulate epileptic activity in brain, adenosine receptor antagonists are pro-convulsant, and adenosinergic mechanisms have been implicated previously in the actions of approved anti-epileptic therapeutics. Here we will review recent literature and describe findings that shed light on mechanistic relationships between ketogenic diets and the purines ATP and adenosine. These emerging mechanisms hold great promise for the effective therapeutic management of epileptic seizures and other neurological conditions.

The ketogenic diet is a high-fat, low-carbohydrate regimen that forces ketone-based rather than glucose-based cellular metabolism. Clinically, maintenance on a ketogenic diet has been proven effective in treating pediatric epilepsy and type II diabetes, and recent basic research provides evidence that ketogenic strategies offer promise in reducing brain injury. Cellular mechanisms hypothesized to be mobilized by ketone metabolism and underlying the success of ketogenic diet therapy, such as reduced reactive oxygen species and increased central adenosine, suggest that the ketolytic metabolism induced by the diet could reduce pain and inflammation. To test the effects of a ketone-based metabolism on pain and inflammation directly, we fed juvenile and adult rats a control diet (standard rodent chow) or ketogenic diet (79% fat) ad libitum for 3-4 weeks. We then quantified hindpaw thermal nociception as a pain measure and complete Freund's adjuvant-induced local hindpaw swelling and plasma extravasation (fluid movement from the vasculature) as inflammation measures. Independent of age, maintenance on a ketogenic diet reduced the peripheral inflammatory response significantly as measured by paw swelling and plasma extravasation. The ketogenic diet also induced significant thermal hypoalgesia independent of age, shown by increased hindpaw withdrawal latency in the hotplate nociception test. Anti-inflammatory and hypoalgesic diet effects were generally more robust in juveniles. The ketogenic diet elevated plasma ketones similarly in both age groups, but caused slowed body growth only in juveniles. These data suggest that applying a ketogenic diet or exploiting cellular mechanisms associated with ketone-based metabolism offers new therapeutic opportunities for controlling pain and peripheral inflammation, and that such a metabolic strategy may offer significant benefits for children and adults.

For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a "retaliatory metabolite." As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor-based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed.

Decreased pH increases extracellular adenosine in CNS regions as diverse as hippocampus and ventral medulla. However, thus far there is no clear consensus whether the critical pH change is a decrease in intracellular and/or extracellular pH. Previously we showed that a decrease in extracellular pH is necessary and a decrease in intracellular pH alone is not sufficient, to increase extracellular adenosine in an acute hippocampal slice preparation. Here we explored further the role of intracellular pH under different synaptic conditions in the hippocampal slice. When synaptic excitability was increased, either during gamma-aminobutyric acid type A receptor blockade in CA1 or after the induction of persistent bursting in CA3, a decrease in intracellular pH alone was now sufficient to: 1) elevate extracellular adenosine concentration, 2) activate adenosine A1 receptors, 3) decrease excitatory synaptic transmission (CA1), and 4) attenuate burst frequency in an in vitro seizure model (CA3). Hippocampal slices obtained from adenosine A1 receptor knockout mice did not exhibit these pH-mediated effects on synaptic transmission, further confirming the role of adenosine acting at the adenosine A1 receptor. Taken together, these data strengthen and add significantly to the evidence outlining a change in pH as an important stimulus influencing extracellular adenosine. In addition, we identify conditions under which intracellular pH plays a dominant role in regulating extracellular adenosine concentrations.

Abnormal neuronal signaling caused by metabolic changes characterizes several neurological disorders, and in some instances metabolic interventions provide therapeutic benefits. Indeed, altering metabolism either by fasting or by maintaining a low-carbohydrate (ketogenic) diet might reduce epileptic seizures and offer neuroprotection in part because the diet increases mitochondrial biogenesis and brain energy levels. Here we focus on a novel hypothesis that a ketogenic diet-induced change in energy metabolism increases levels of ATP and adenosine, purines that are critically involved in neuron-glia interactions, neuromodulation and synaptic plasticity. Enhancing brain bioenergetics (ATP) and increasing levels of adenosine, an endogenous anticonvulsant and neuroprotective molecule, might help with understanding and treating a variety of neurological disorders.

There is significant interest in in vivo synaptic plasticity in mice due to the many relevant genetic mutants now available. Nevertheless, use of in vivo models remains limited. To date long-term potentiation (LTP) has been studied infrequently, and long-term depression (LTD) has not been characterized in the mouse in vivo. Herein we describe protocols and improved methodologies we developed to record hippocampal synaptic plasticity reliably from the dentate gyrus of the awake freely behaving mouse. Seven days prior to recording, we implanted microelectrodes encapsulated within a lightweight, low profile head stage assembly. On the day of recording, we induced either LTP or LTD in the awake freely behaving animal, and monitored subsequent changes in population spike amplitude for at least 24h. Using this protocol we attained 80% success in inducing and maintaining either LTP or LTD. Recording from a chronic implant using this improved methodology is best suited to reveal naturally occurring brain activity and avoids both acute effects of local electrode insertion and drifts in neuronal excitability associated with anesthesia. Ultimately a reliable freely behaving mouse model of bi-directional synaptic plasticity is invaluable for full characterization of genetic models of disease states and manipulations of the mechanisms implicated in learning and memory.

In addition to affecting respiration and vascular tone, deviations from normal CO(2) alter pH, consciousness, and seizure propensity. Outside the brainstem, however, the mechanisms by which CO(2) levels modify neuronal function are unknown. In the hippocampal slice preparation, increasing CO(2), and thus decreasing pH, increased the extracellular concentration of the endogenous neuromodulator adenosine and inhibited excitatory synaptic transmission. These effects involve adenosine A(1) and ATP receptors and depend on decreased extracellular pH. In contrast, decreasing CO(2) levels reduced extracellular adenosine concentration and increased neuronal excitability via adenosine A(1) receptors, ATP receptors, and ecto-ATPase. Based on these studies, we propose that CO(2)-induced changes in neuronal function arise from a pH-dependent modulation of adenosine and ATP levels. These findings demonstrate a mechanism for the bidirectional effects of CO(2) on neuronal excitability in the forebrain.

Using mice with a targeted disruption of the adenosine A1 receptor (A1R), we examined the role of A1Rs in hippocampal long-term potentiation (LTP), long-term depression (LTD), and memory formation. Recordings from the Shaffer collateral-CA1 pathway of hippocampal slices from adult mice showed no differences between theta burst and tetanic stimulation-induced LTP in adenosine A1 receptor knockout (A1R-/-), heterozygote (A1R+/-), and wildtype (A1R+/+) mice. However, paired pulse facilitation was impaired significantly in A1R-/- slices as compared to A1R+/+ slices. LTD in the CA1 region was unaffected by the genetic manipulation. The three genotypes showed similar memory acquisition patterns when assessed for spatial reference and working memory in the Morris water maze tasks at 9 months of age. However, 10 months later A1R-/- mice showed some deficits in the 6-arm radial tunnel maze test. The latter appeared, however, not due to memory deficits but to decreased habituation to the test environment. Taken together, we observe normal spatial learning and memory and hippocampal CA1 synaptic plasticity in adult adenosine A1R knockout mice, but find modifications in arousal-related processes, including habituation, in this knockout model.

Adenosine's role in the nervous system is multifaceted. As the core molecule of adenosine triphosphate (ATP), adenosine exists in equilibrium with the adenine nucleotide pool and contributes to cellular energy charge, a quantification of relative amounts of available ATP, ADP, AMP and adenosine. Beyond participating in overall energy balance and thus in maintaining cellular homeostasis, adenosine critically influences dynamic signaling in the nervous system. In particular, adenosine has an effect on and is affected by excitatory synaptic transmission. This report describes the ubiquitous nature of adenosine's influence, outlines specific scenarios of clinical import and highlights emerging knowledge about the regulation of adenosine.

Adenosine and its receptors have been the topic of many recent reviews. These reviews provide a good summary of much of the relevant literature--including the older literature. We have, therefore, chosen to focus the present review on the insights gained from recent studies on genetically modified mice, particularly with respect to the function of adenosine receptors and their potential as therapeutic targets. The information gained from studies of drug effects is discussed in this context, and discrepancies between genetic and pharmacological results are highlighted.