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Addiction is a brain disease, and current diagnostic criteria for substance use disorders (SUDs) are qualitative. Nevertheless, scientific advances are beginning to characterize neurobiological domains. Combining multiple units of measure may provide an opportunity to deconstruct the heterogeneities of a SUD and define endophenotypes by using peripheral biospecimens. There are several recent examples of potential biomarker types that can be examined, together with their categorical applications for SUDs. We propose that, in conjunction with rapidly advancing statistical and mathematical modeling techniques, there is now a unique opportunity for the discovery of composite biomarkers within specific domains of addiction; these may lay the foundation for future biomarker qualification, with important implications for drug development and medical care., (Published by Elsevier Ltd.)

There has been significant progress in personalized drug development. In large part, this has taken place in the oncology field and been due to the ability of researchers/clinicians to discover and develop novel drug development tools (DDTs), such as biomarkers. In cancer treatment research, biomarkers have permitted a more accurate pathophysiological characterization of an individual patient, and have enabled practitioners to target mechanistically the right drug, to the right patient, at the right time. Similar to cancer, patients with substance use disorders (SUDs) present clinically with heterogeneous symptomatology and respond variably to therapeutic interventions. If comparable biomarkers could be identified and developed for SUDs, significant diagnostic and therapeutic advances could be made. In this review, we highlight current opportunities and difficulties pertaining to the identification and development of biomarkers for SUDs. We focus on cocaine dependence as an example. Putative diagnostic, pharmacodynamic (PD), and predictive biomarkers for cocaine dependence are discussed across a range of methodological approaches. A possible cocaine-dependent clinical outcome assessment (COA)--another type of defined DDT--is also discussed. At present, biomarkers for cocaine dependence are in their infancy. Much additional research will be needed to identify, validate, and qualify these putative tools prior to their potential use for medications development and/or application to clinical practice. However, with a large unmet medical need and an estimated market size of several hundred million dollars per year, if developed, biomarkers for cocaine dependence will hold tremendous value to both industry and public health.

One way to enhance therapeutic development is through the identification and development of evaluative tools such as biomarkers. This review focuses on putative diagnostic, pharmacodynamic, and predictive biomarkers for smoking cessation. These types of biomarkers may be used to more accurately diagnose a disease, personalize treatment, identify novel targets for drug discovery, and enhance the efficiency of drug development. Promising biomarkers are presented across a range of approaches including metabolism, genetics, and neuroimaging. A preclinical viewpoint is also offered, as are analytical considerations and a regulatory perspective summarizing a pathway toward biomarker qualification.

Excessive glutamatergic neurotransmission is considered an underlying factor of epilepsy. Energy-dependent glutamate transporters clear extracellular glutamate to limit neuronal excitability. Evidence suggests that reduced expression and/or activity of glutamate transporters contribute to hyperexcitability and progressive seizure activity in rats. By comparison, treatment with the anticonvulsant ketogenic diet (KD) results in increased mRNA expression of the neuronal glutamate transporter EAAC1, elevated energy reserves, and an increased resistance to seizures in rats. The goal of the current study was to determine whether the expression and/or re-uptake activity of glutamate transporters were elevated in hippocampal tissue of rats after KD treatment. Rats were fed either a ketogenic- or control diet for 4-5 weeks. Western blot analysis showed that protein levels of EAAC1, GLT-1 and GLAST glutamate transporters were not changed in hippocampus, cerebral cortex, or cerebellum after KD. Electron microscopic evidence indicated that the KD did not affect hippocampal EAAC1 distribution. In addition, the re-uptake activity of (3)H-glutamate into hippocampal proteoliposomes was similar in both KD and control tissue extracts. These multiple studies suggest that the anticonvulsant nature of the KD does not stem from enhanced glutamate re-uptake.

The ketogenic diet (KD) is a broadly effective treatment for medically refractory epilepsy. Despite nearly a century of use, the mechanisms underlying its clinical efficacy remain unknown. In this review, we present one intersecting view of how the KD may exert its anticonvulsant activity against the backdrop of several seemingly disparate mechanistic theories. We summarize key insights gleaned from experimental and clinical studies of the KD, and focus particular attention on the role that ketone bodies, fatty acids, and limited glucose may play in seizure control. Chronic ketosis is anticipated to modify the tricarboxcylic acid cycle to increase GABA synthesis in brain, limit reactive oxygen species (ROS) generation, and boost energy production in brain tissue. Among several direct neuro-inhibitory actions, polyunsaturated fatty acids increased after KD induce the expression of neuronal uncoupling proteins (UCPs), a collective up-regulation of numerous energy metabolism genes, and mitochondrial biogenesis. These effects further limit ROS generation and increase energy production. As a result of limited glucose and enhanced oxidative phosphorylation, reduced glycolytic flux is hypothesized to activate metabolic K(ATP) channels and hyperpolarize neurons and/or glia. Although it is unlikely that a single mechanism, however well substantiated, will explain all of the diet's clinical benefits, these diverse, coordinated changes seem poised to stabilize synaptic function and increase the resistance to seizures throughout the brain.

Objective: The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect., Methods: Microarrays were used to define patterns of gene expression in the hippocampus of rats fed a KD or control diet for 3 weeks. Hippocampi from control- and KD-fed rats were also compared for the number of mitochondrial profiles in electron micrographs, the levels of selected energy metabolites and enzyme activities, and the effect of low glucose on synaptic transmission., Results: Most striking was a coordinated upregulation of all (n = 34) differentially regulated transcripts encoding energy metabolism enzymes and 39 of 42 transcripts encoding mitochondrial proteins, which was accompanied by an increased number of mitochondrial profiles, a higher phosphocreatine/creatine ratio, elevated glutamate levels, and decreased glycogen levels. Consistent with increased energy reserves, synaptic transmission in hippocampal slices from KD-fed animals was resistant to low glucose., Interpretation: These data show that a calorie-restricted KD enhances brain metabolism. We propose an anticonvulsant mechanism of the KD involving mitochondrial biogenesis leading to enhanced alternative energy stores.

We investigated group II metabotropic glutamate receptor (mGluR) modulation of glutamatergic input onto hilar-border interneurones and its regulation of feedback inhibition in the dentate gyrus. Selective activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) depressed mossy fibre (MF)-evoked excitatory drive to these interneurones with significantly greater depression in juvenile than adult rats. During 20 Hz MF stimulus trains, EPSCs became depressed. Depression during the early, but not later part of the train was significantly greater in juvenile than adult rats and was blocked by the mGluR antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). In dentate granule cells from juvenile rats polysynaptic feedback IPSCs, but not monosynaptic IPSCs, were strongly suppressed by DCG-IV. DCG-IV also suppressed feedback inhibition of perforant path-evoked population spikes. In contrast, in adult animals DCG-IV did not significantly depress feedback inhibition. During 20 Hz stimulus trains in juvenile animals the summation of polysynaptic, but not monosynaptic IPSCs was suppressed by synaptically activated group II mGluRs. Blockade of these mGluRs with LY341495 significantly increased the area and duration of the summated IPSC, causing greater feedback inhibition of granule cell firing. In contrast, in adult animals LY341495 did not alter feedback inhibition following the stimulus train. These findings indicate that group II mGluRs modulate excitatory drive to interneurones in a developmentally regulated manner and thereby modulate feedback inhibition in the dentate gyrus.

Metabotropic glutamate receptor (mGluR)-mediated inhibition within the dentate gyrus is altered after epilepsy. Whether these changes occur during the developmental period of the disease (i.e., the latent period) has not yet been investigated. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the lateral (LPP) and medial perforant path (MPP) simultaneously in adult mouse hippocampal slices 3-9 days after pilocarpine (PILO)-induced status epilepticus. Genetically manipulated mice (mGluR8 knockout and mGluR4/8 double knockout) and pharmacologically selective agonists were used to identify specific mGluR subtypes affected after PILO. Pharmacological activation of mGluR7 by L-AP4 in both wild-type and mGluR4/8 double knockout mice selectively reduced fEPSPs in the MPP, but not LPP, and this level of inhibition was significantly reduced 3-9 days after PILO-induced SE. Activation of mGluR2/3 reversibly depressed the fEPSP slopes in both the MPP and LPP, but no alterations were noted after PILO. mGluR8 activation selectively inhibited evoked responses in the LPP, but not in the MPP, and this level of inhibition did not change after PILO treatment. These data suggest that reduced presynaptic inhibition mediated by mGluR7, but not mGluR2/3 or mGluR8, may play a role during the latent period in generating hyperexcitability in the dentate and thereby contribute to epileptogenesis.

Purpose: The ketogenic diet (KD) is an effective treatment for intractable epilepsy. However, little is known about its underlying mechanisms., Methods: In this study, in vivo extracellular field responses to angular bundle stimulation were recorded in the dentate gyrus of Sprague-Dawley rats fed one of three diets: ketogenic calorie-restricted (KCR), normal calorie-restricted (NCR), or normal ad libitum (NAL). Input/output curves and paired-pulse relations were used to assess network excitability. A maximal dentate activation (MDA) protocol was used to measure electrographic seizure threshold and duration., Results: Animals fed calorie-restricted (CR) diets exhibited greater paired-pulse inhibition, an elevated MDA threshold, and an absence of spreading depression-like events compared with ad libitum-fed controls. In the MDA model of epileptogenesis, the rate of increase in electrographic seizure duration after repeated stimuli was markedly reduced in KCR-fed animals compared with NCR- and NAL-fed controls., Conclusions: These data suggest that CR, by itself, can be anticonvulsant, and treatment with a KCR diet may be both anticonvulsant and antiepileptogenic.

The ketogenic diet (KD) is a high-fat, low-carbohydrate, adequate-protein diet that has been used for more than eight decades for the treatment of refractory epilepsy in children. Despite this long history, the mechanisms by which the KD exerts its anti-seizure action are not fully understood. Questions remain regarding several aspects of KD action, including its effects on brain biochemistry and energetics, neuronal membrane function and cellular network behavior. With the explosion of the KD use in the last 10 years, it is now imperative that we understand these factors in greater detail, in order to optimize the formulation, administration and fine-tuning of the diet. This review discusses what is known and what remains to be learned about the KD, with emphasis on clinical questions that can be approached in the laboratory. We encourage scientists with a primary interest in nutritional neuroscience to join with those of us in the epilepsy research community to address these urgent questions, for the benefit of children ravaged by intractable seizures.

The present study was designed to evaluate the anticonvulsant effects of a high-fat ketogenic diet (KD) in rats. Animals were maintained on one of four experimental diets: (1) calorie-restricted ketogenic (KCR); (2) calorie-restricted normal (NCR); (3) ad libitum ketogenic (KAL); or (4) ad libitum normal (NAL). The calorie-restricted diets were fed in quantities such that they were calorically equivalent. All animals began diet treatment at age P37 and each was subjected to one of five chemically-induced seizure tests: bicuculline (BIC; s.c.), picrotoxin (PIC; s.c.), kainate (KA, i.p. or s.c.) and gamma-butyrolactone (GBL, i.p.), strychnine (s.c.). Bipolar epidural electrodes were implanted under ketamine/xylazine anesthesia to permit recording the spike and wave discharges (SWD) characteristic of electroencephalograms during absence seizures. Ketonemia was assayed by measuring blood levels of beta-hydroxybutyrate (BHB) spectrophotometrically prior to induction of seizures in each experiment. Animals fed ketogenic diets (i.e. either calorie restricted or ad libitum) exhibited greater blood levels of BHB compared to control groups. Seizure results show that treatment with a KD: (1) reduced the incidence of bicuculline-induced convulsions; (2) diminished the number of picrotoxin-induced seizures (KCR group only); (3) increased latency to GBL-induced SWD and reduced both the number and duration of SWD; but (4) conferred no protection from strychnine-induced seizures; and (5) made KA-induced seizures more severe. Together these results indicate a spectrum of anticonvulsant action for the KD in rats that includes threshold seizures induced via GABA receptors (BIC, PIC, GBL) but not those induced at glycine (strychnine) or the KA-subclass of glutamate receptors. Uniquely, the KD is the only treatment described that protects against both convulsive and non-convulsive (absence) seizures in rats.

Purpose: The purpose of this study was to measure quantitatively the effectiveness of the ketogenic diet (KD) in comparison to two clinically important anticonvulsant drugs (AEDs), valproic acid (VPA) and phenytoin (PHT), and to evaluate possible associated neurotoxicity., Methods: Rats were maintained on either a calorie-restricted, KD or calorie-restricted, rodent-chow diet for 3-5 weeks, after which neurobehavioral and seizure testing was completed. AEDs (either VPA or PHT) were injected acutely at the time to peak effect before neurotoxic and seizure assessment. Seizures were induced by timed infusion of pentylenetetrazole (PTZ) and maximal electroshock (MES)., Results: VPA protected from both MES- and PTZ-induced seizures, whereas the KD only elevated PTZ seizure threshold; PHT only attenuated MES-induced seizures. The KD was as effective as a high dose of VPA (i.e., 300 mg/kg) and combined treatment (i.e., KD + VPA) showed an additive increase in PTZ seizure threshold. No observed neurobehavioral deficits were associated with either diet treatment; however, drug-related side effects were noted with high doses of either VPA or PHT., Conclusions: These data suggest that the KD ranks among VPA and PHT as an effective treatment for seizures, without observed drug-associated neurobehavioral contraindications. In combination with AEDs, our results indicate that the KD plus VPA work synergistically to increase seizure threshold, whereas the KD plus PHT may be complementary, elevating seizure threshold (KD) and reducing seizure severity (PHT). These findings may provide insights into future directions for rational polytherapy; however, it is important to be aware that the KD has been shown to elevate VPA-induced hepatotoxicity.

The purpose of these experiments was to determine whether a ketogenic diet previously shown to elevate seizure threshold also reduced seizure severity. Seizure threshold was tested by intravenous infusion of pentylenetetrazole (PTZ) whereas seizure severity was determined from measuring the hindlimb extension to flexion (E/F) ratio after seizures were evoked by maximal electroshock stimulation (MES). Surprisingly, seizures evoked by MES were more severe in animals fed a calorie-restricted ketogenic diet. Controls fed an isocaloric, calorie-restricted normal diet also exhibited more severe seizures than did animals fed the same diet ad libitum. When seizure threshold was evaluated in the same animals, those animals fed a calorie-restricted ketogenic diet exhibited a significant increase in seizure resistance compared to animals fed a ketogenic diet ad libitum, a calorie-restricted normal diet or a normal diet ad libitum. These findings suggest that both the amount and type of food affect seizures in rats and show that diet-related seizure protection depends upon the method by which seizures are provoked.

The present study was designed to establish a dose-response relationship for the efficacy of the ketogenic diet (KD). Sprague-Dawley rats were fed ketogenic diets containing varying ratios of fats; (carbohydrates + proteins) whereas control animals were fed rodent chow. Unless otherwise indicated, all animals were fed calorie-restricted, isocaloric diets beginning at P37 and ketonemia, seizure threshold and neurotoxic effects were determined. Despite being provided isocaloric quantities, animals fed lower ketogenic ratios gained weight relative to those fed diets having greater proportions of fats. A significantly increased metabolic rate was noted for animals fed a high-fat diet, suggesting a basis for the weight differences. Results also showed that the animals fed calorie-restricted high-fat diets exhibited significant ketonemia and protection from pentylenetetrazole (PTZ)-induced seizures. There were no detectable neurotoxic effects for any diet group. For animals of the same age, there was no correlation between beta-hydroxybutyrate (beta-OHB) and seizure threshold. These findings suggest that beta-OHB is not directly involved in the anticonvulsant mechanism of the diet. Also, data presented here show that the conventional 4:1 ketogenic diet does not confer the greatest level of seizure protection. We conclude that a 6:1 ketogenic diet, which shows no evidence of neurotoxicity, may be maximally efficacious in rats.

Previous work has identified several criteria that may be important in determining the efficacy of the ketogenic diet as a treatment for intractable epilepsy in children. The present study was designed to investigate the influence of four major variables on seizure threshold, i.e. ketogenic ratio, body weight, age at diet onset and beta-hydroxybutyrate in rats. Path analysis was used to statistically model and quantify the causal relationships among variables. Results indicate that seizure threshold was significantly elevated with increasing ketogenic ratios (i.e. more fats vs. carbohydrates and proteins) and decreasing weight. Conversely, age at diet onset and plasma levels of beta-OHB showed no causal relation to seizure resistance. These results suggest that the efficacy of the ketogenic diet is independent of the level of ketonemia but is markedly influenced by diet and growth., (Copyright 1999 S. Karger AG, Basel)

The present study was designed to evaluate the effects of age on the efficacy of the ketogenic diet in suppressing seizures evoked by tail-vein infusion of pentylenetetrazole (PTZ). Male rats of various ages were divided into three groups and fed one of three diets: (1) a calorie-restricted ketogenic diet, (2) a calorie-restricted normal (rodent chow) diet, or (3) a normal diet, ad libitum. After animals had been on experimental or control diets for more than 20 days, seizure threshold and blood levels of beta-hydroxybutyrate (beta-OHB) were determined. Animals fed a ketogenic diet exhibited significant elevations in levels of beta-OHB and seizure resistance compared to animals fed either a calorie-restricted normal diet or a normal diet, ad libitum. The levels of beta-OHB and seizure resistance were greatest for young pups. A surprising finding was that young animals fed a calorie-restricted rodent chow diet exhibited a significantly increased resistance to seizures compared to those fed the same diet, ad libitum. Results presented here demonstrate that the ketogenic diet produces the highest levels of ketonemia and seizure threshold in young animals. Collectively, these data suggest that age and caloric restriction are important considerations for implementing the ketogenic diet.

Purpose: The purpose of this study was to test the hypothesis that a ketogenic diet would increase the resistance of rats to pentylenetetrazole (PTZ)-induced seizures and to understand the relation of ketonemia to seizure resistance., Methods: A freely consumed, high-fat (ketogenic) diet was administered to male Sprague-Dawley rats for 5-10 weeks, while control animals were fed either rodent chow or a high-carbohydrate diet. Ketonemia was measured as plasma levels of beta-hydroxybutyric acid (beta-OHB). Seizures were induced by tail-vein infusion of pentylenetetrazole., Results: The ketogenic diet produced a highly significant (p<0.01) increase in beta-OHB levels within 5 days. Induction of seizures by PTZ 35 days after animals were placed on their respective diets showed that ketogenic animals had a significantly (p<0.01) increased threshold for seizure induction compared with those fed an isocaloric diet of either high-carbohydrate or normal rodent chow. Ketogenic animals did not exhibit increased seizure severity relative to controls, despite receiving consistently higher doses of PTZ., Conclusions: The ketogenic diet resulted in an increased seizure threshold, confirming the hypothesis, and seizure threshold was found to be a direct function of the level of ketonemia.