Isolation of the murine Glut1 deficient thalamocortical circuit: wavelet characterization and reverse glucose dependence of low and gamma frequency oscillations.

Glucose represents the principal brain energy source. Thus, not unexpectedly, genetic glucose transporter 1 (Glut1) deficiency (G1D) manifests with encephalopathy. G1D seizures, which constitute a prominent disease manifestation, often prove refractory to medications but may respond to therapeutic diets. These seizures are associated with aberrant thalamocortical oscillations as inferred from human electroencephalography and functional imaging. Mouse electrophysiological recordings indicate that inhibitory neuron failure in thalamus and cortex underlies these abnormalities. This provides the motivation to develop a neural circuit testbed to characterize the mechanisms of thalamocortical synchronization and the effects of known or novel interventions. To this end, we used mouse thalamocortical slices on multielectrode arrays and characterized spontaneous low frequency oscillations and less frequent 30-50 Hz or gamma oscillations under near-physiological bath glucose concentration. Using the cortical recordings from layer IV among other regions recorded, we quantified oscillation epochs via an automated wavelet-based algorithm. This method proved analytically superior to power spectral density, short-time Fourier transform or amplitude-threshold detection. As expected from human observations, increased bath glucose reduced the lower frequency oscillations while augmenting the gamma oscillations, likely reflecting strengthened inhibitory neuron activity, and thus decreasing the low:high frequency ratio (LHR). This approach provides an ex vivo method for the evaluation of mechanisms, fuels, and pharmacological agents in a crucial G1D epileptogenic circuit.

Maintenance of pig brain function under extracorporeal pulsatile circulatory control (EPCC).

Selective vascular access to the brain is desirable in metabolic tracer, pharmacological and other studies aimed to characterize neural properties in isolation from somatic influences from chest, abdomen or limbs. However, current methods for artificial control of cerebral circulation can abolish pulsatility-dependent vascular signaling or neural network phenomena such as the electrocorticogram even while preserving individual neuronal activity. Thus, we set out to mechanically render cerebral hemodynamics fully regulable to replicate or modify native pig brain perfusion. To this end, blood flow to the head was surgically separated from the systemic circulation and full extracorporeal pulsatile circulatory control (EPCC) was delivered via a modified aorta or brachiocephalic artery. This control relied on a computerized algorithm that maintained, for several hours, blood pressure, flow and pulsatility at near-native values individually measured before EPCC. Continuous electrocorticography and brain depth electrode recordings were used to evaluate brain activity relative to the standard offered by awake human electrocorticography. Under EPCC, this activity remained unaltered or minimally perturbed compared to the native circulation state, as did cerebral oxygenation, pressure, temperature and microscopic structure. Thus, our approach enables the study of neural activity and its circulatory manipulation in independence of most of the rest of the organism.

Isolation of the murine Glut1 deficient thalamocortical circuit: wavelet characterization and reverse glucose dependence of low and gamma frequency oscillations.

Glucose represents the principal brain energy source. Thus, not unexpectedly, genetic glucose transporter 1 (Glut1) deficiency (G1D) manifests with encephalopathy. G1D seizures, which constitute a prominent disease manifestation, often prove refractory to medications but may respond to therapeutic diets. These seizures are associated with aberrant thalamocortical oscillations as inferred from human electroencephalography and functional imaging. Mouse electrophysiological recordings indicate that inhibitory neuron failure in thalamus and cortex underlies these abnormalities. This provides the motivation to develop a neural circuit testbed to characterize the mechanisms of thalamocortical synchronization and the effects of known or novel interventions. To this end, we used mouse thalamocortical slices on multielectrode arrays and characterized spontaneous low frequency oscillations and less frequent 30-50 Hz or gamma oscillations under near-physiological bath glucose concentration. Using the cortical recordings from layer IV, we quantified oscillation epochs via an automated wavelet-based algorithm. This method proved analytically superior to power spectral density, short-time Fourier transform or amplitude-threshold detection. As expected from human observations, increased bath glucose reduced the lower frequency oscillations while augmenting the gamma oscillations, likely reflecting strengthened inhibitory neuron activity. This approach provides an ex vivo method for the evaluation of mechanisms, fuels, and pharmacological agents in a crucial G1D epileptogenic circuit.

Identification of Glucose Transport Modulators In Vitro and Method for Their Deep Learning Neural Network Behavioral Evaluation in Glucose Transporter 1-Deficient Mice.

Metabolic flux augmentation via glucose transport activation may be desirable in glucose transporter 1 (Glut1) deficiency syndrome (G1D) and dementia, whereas suppression might prove useful in cancer. Using lung adenocarcinoma cells that predominantly express Glut1 relative to other glucose transporters, we screened 9,646 compounds for effects on the accumulation of an extracellularly applied fluorescent glucose analog. Five drugs currently prescribed for unrelated indications or preclinically characterized robustly enhanced intracellular fluorescence. Additionally identified were 37 novel activating and nine inhibitory compounds lacking previous biologic characterization. Because few glucose-related mechanistic or pharmacological studies were available for these compounds, we developed a method to quantify G1D mouse behavior to infer potential therapeutic value. To this end, we designed a five-track apparatus to record and evaluate spontaneous locomotion videos. We applied this to a G1D mouse model that replicates the ataxia and other manifestations cardinal to the human disorder. Because the first two drugs that we examined in this manner (baclofen and acetazolamide) exerted various impacts on several gait aspects, we used deep learning neural networks to more comprehensively assess drug effects. Using this method, 49 locomotor parameters differentiated G1D from control mice. Thus, we used parameter modifiability to quantify efficacy on gait. We tested this by measuring the effects of saline as control and glucose as G1D therapy. The results indicate that this in vivo approach can estimate preclinical suitability from the perspective of G1D locomotion. This justifies the use of this method to evaluate our drugs or other interventions and sort candidates for further investigation. SIGNIFICANCE STATEMENT: There are few or no activators and few clinical inhibitors of glucose transport. Using Glut1-rich cells exposed to a glucose analog, we identified, in highthroughput fashion, a series of novel modulators. Some were drugs used to modify unrelated processes and some represented large but little studied chemical compound families. To facilitate their preclinical efficacy characterization regardless of potential mechanism of action, we developed a gait testing platform for deep learning neural network analysis of drug impact on Glut1-deficient mouse locomotion.

Red blood cells as glucose carriers to the human brain: Modulation of cerebral activity by erythrocyte exchange transfusion in Glut1 deficiency (G1D).

Red blood cells circulating through the brain are briefly but closely apposed to the capillary endothelium. We hypothesized that this contact provides a nearly direct pathway for metabolic substrate transfer to neural cells that complements the better characterized plasma to endothelium transfer. While brain function is considered independent of normal fluctuations in blood glucose concentration, this is not borne out by persons with glucose transporter I (GLUT1) deficiency (G1D). In them, encephalopathy is often ameliorated by meal or carbohydrate administration, and this enabled us to test our hypothesis: Since red blood cells contain glucose, and since the red cells of G1D individuals are also deficient in GLUT1, replacing them with normal donor cells via exchange transfusion could augment erythrocyte to neural cell glucose transport via mass action in the setting of unaltered erythrocyte count or plasma glucose abundance. This motivated us to perform red blood cell exchange in 3 G1D persons. There were rapid, favorable and unprecedented changes in cognitive, electroencephalographic and quality-of-life measures. The hypothesized transfer mechanism was further substantiated by in vitro measurement of direct erythrocyte to endothelial cell glucose flux. The results also indicate that the adult intellect is capable of significant enhancement without deliberate practice. ClinicalTrials.gov registration: NCT04137692 https://clinicaltrials.gov/ct2/show/NCT04137692.

Metabolic modulation of synaptic failure and thalamocortical hypersynchronization with preserved consciousness in Glut1 deficiency.

Individuals with glucose transporter type I deficiency (G1D) habitually experience nutrient-responsive epilepsy associated with decreased brain glucose. However, the mechanistic association between blood glucose concentration and brain excitability in the context of G1D remains to be elucidated. Electroencephalography (EEG) in G1D individuals revealed nutrition time-dependent seizure oscillations often associated with preserved volition despite electrographic generalization and uniform average oscillation duration and periodicity, suggesting increased facilitation of an underlying neural loop circuit. Nonlinear EEG ictal source localization analysis and simultaneous EEG/functional magnetic resonance imaging converged on the thalamus-sensorimotor cortex as one potential circuit, and 18F-deoxyglucose positron emission tomography (18F-DG-PET) illustrated decreased glucose accumulation in this circuit. This pattern, reflected in a decreased thalamic to striatal 18F signal ratio, can aid with the PET imaging diagnosis of the disorder, whereas the absence of noticeable ictal behavioral changes challenges the postulated requirement for normal thalamocortical activity during consciousness. In G1D mice, 18F-DG-PET and mass spectrometry also revealed decreased brain glucose and glycogen, but preserved tricarboxylic acid cycle intermediates, indicating no overall energy metabolism failure. In brain slices from these animals, synaptic inhibition of cortical pyramidal neurons and thalamic relay neurons was decreased, and neuronal disinhibition was mitigated by metabolic sources of carbon; tonic-clonic seizures were also suppressed by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition. These results pose G1D as a thalamocortical synaptic disinhibition disease associated with increased glucose-dependent neuronal excitability, possibly in relation to reduced glycogen. Together with findings in other metabolic defects, inhibitory neuron dysfunction is emerging as a modulable mechanism of hyperexcitability.

Recording of pig neuronal activity in the comparative context of the awake human brain.

Gyriform mammals display neurophysiological and neural network activity that other species exhibit only in rudimentary or dissimilar form. However, neural recordings from large mammals such as the pig can be anatomically hindered and pharmacologically suppressed by anesthetics. This curtails comparative inferences. To mitigate these limitations, we set out to modify electrocorticography, intracerebral depth and intracortical recording methods to study the anesthetized pig. In the process, we found that common forms of infused anesthesia such as pentobarbital or midazolam can be neurophysiologic suppressants acting in dose-independent fashion relative to anesthetic dose or brain concentration. Further, we corroborated that standard laboratory conditions may impose electrical interference with specific neural signals. We thus aimed to safeguard neural network integrity and recording fidelity by developing surgical, anesthesia and noise reduction methods and by working inside a newly designed Faraday cage, and evaluated this from the point of view of neurophysiological power spectral density and coherence analyses. We also utilized novel silicon carbide electrodes to minimize mechanical disruption of single-neuron activity. These methods allowed for the preservation of native neurophysiological activity for several hours. Pig electrocorticography recordings were essentially indistinguishable from awake human recordings except for the small segment of electrical activity associated with vision in conscious persons. In addition, single-neuron and paired-pulse stimulation recordings were feasible simultaneously with electrocorticography and depth electrode recordings. The spontaneous and stimulus-elicited neuronal activities thus surveyed can be recorded with a degree of precision similar to that achievable in rodent or any other animal studies and prove as informative as unperturbed human electrocorticography.

Brain metabolism modulates neuronal excitability in a mouse model of pyruvate dehydrogenase deficiency.

Glucose is the ultimate substrate for most brain activities that use carbon, including synthesis of the neurotransmitters glutamate and γ-aminobutyric acid via mitochondrial tricarboxylic acid (TCA) cycle. Brain metabolism and neuronal excitability are thus interdependent. However, the principles that govern their relationship are not always intuitive because heritable defects of brain glucose metabolism are associated with the paradoxical coexistence, in the same individual, of episodic neuronal hyperexcitation (seizures) with reduced basal cerebral electrical activity. One such prototypic disorder is pyruvate dehydrogenase (PDH) deficiency (PDHD). PDH is central to metabolism because it steers most of the glucose-derived flux into the TCA cycle. To better understand the pathophysiology of PDHD, we generated mice with brain-specific reduced PDH activity that paralleled salient human disease features, including cerebral hypotrophy, decreased amplitude electroencephalogram (EEG), and epilepsy. The mice exhibited reductions in cerebral TCA cycle flux, glutamate content, spontaneous, and electrically evoked in vivo cortical field potentials and gamma EEG oscillation amplitude. Episodic decreases in gamma oscillations preceded most epileptiform discharges, facilitating their prediction. Fast-spiking neuron excitability was decreased in brain slices, contributing to in vivo action potential burst prolongation after whisker pad stimulation. These features were partially reversed after systemic administration of acetate, which augmented cerebral TCA cycle flux, glutamate-dependent synaptic transmission, inhibition and gamma oscillations, and reduced epileptiform discharge duration. Thus, our results suggest that dysfunctional excitability in PDHD is consequent to reduced oxidative flux, which leads to decreased neuronal activation and impaired inhibition, and can be mitigated by an alternative metabolic substrate.

Triheptanoin for glucose transporter type I deficiency (G1D): modulation of human ictogenesis, cerebral metabolic rate, and cognitive indices by a food supplement.

IMPORTANCE: Disorders of brain metabolism are multiform in their mechanisms and manifestations, many of which remain insufficiently understood and are thus similarly treated. Glucose transporter type I deficiency (G1D) is commonly associated with seizures and with electrographic spike-waves. The G1D syndrome has long been attributed to energy (ie, adenosine triphosphate synthetic) failure such as that consequent to tricarboxylic acid (TCA) cycle intermediate depletion. Indeed, glucose and other substrates generate TCAs via anaplerosis. However, TCAs are preserved in murine G1D, rendering energy-failure inferences premature and suggesting a different hypothesis, also grounded on our work, that consumption of alternate TCA precursors is stimulated and may be detrimental. Second, common ketogenic diets lead to a therapeutically counterintuitive reduction in blood glucose available to the G1D brain and prove ineffective in one-third of patients. OBJECTIVE: To identify the most helpful outcomes for treatment evaluation and to uphold (rather than diminish) blood glucose concentration and stimulate the TCA cycle, including anaplerosis, in G1D using the medium-chain, food-grade triglyceride triheptanoin. DESIGN, SETTING, AND PARTICIPANTS: Unsponsored, open-label cases series conducted in an academic setting. Fourteen children and adults with G1D who were not receiving a ketogenic diet were selected on a first-come, first-enrolled basis. INTERVENTION: Supplementation of the regular diet with food-grade triheptanoin. MAIN OUTCOMES AND MEASURES: First, we show that, regardless of electroencephalographic spike-waves, most seizures are rarely visible, such that perceptions by patients or others are inadequate for treatment evaluation. Thus, we used quantitative electroencephalographic, neuropsychological, blood analytical, and magnetic resonance imaging cerebral metabolic rate measurements. RESULTS: One participant (7%) did not manifest spike-waves; however, spike-waves promptly decreased by 70% (P = .001) in the other participants after consumption of triheptanoin. In addition, the neuropsychological performance and cerebral metabolic rate increased in most patients. Eleven patients (78%) had no adverse effects after prolonged use of triheptanoin. Three patients (21%) experienced gastrointestinal symptoms, and 1 (7%) discontinued the use of triheptanoin. CONCLUSIONS AND RELEVANCE: Triheptanoin can favorably influence cardinal aspects of neural function in G1D. In addition, our outcome measures constitute an important framework for the evaluation of therapies for encephalopathies associated with impaired intermediary metabolism.

Modeling of brain metabolism and pyruvate compartmentation using (13)C NMR in vivo: caution required.

Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,6-(13)C2]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ. Furthermore, the models included the capacity to analyze multiplets arising from (13)C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-(13)C2]glucose metabolism. Four analyses were used, two comparing the effect of different pyruvate enrichment in glia and neurons, and two for determining the effect of multiplets present in the data. When fit independently, the enrichment in glial pyruvate was less than in neurons. In the absence of multiplets, fit quality and parameter values were typical of those in the literature, whereas the multiplet curves were not modeled well. This prompted the use of robust statistical analysis (the Kolmogorov-Smirnov test of goodness of fit) to determine whether individual curves were modeled appropriately. At least 50% of the curves in each experiment were considered poorly fit. It was concluded that the model does not include all metabolic features required to analyze the data.

Surgical knot integrity: effect of suture type and caliber, and level of residency training.

OBJECTIVE: To evaluate if suture type and caliber or level of residency training affects strength and mode of failure of surgical knots. DESIGN: All residents in an obstetrics and gynecology training program were invited to tie knots on a bench model using 2 calibers (0 and 3-0) of 2 types of surgical suture (polyglactin 910 and polydioxanone). The failure load, mode of failure, and loop lengths of the knots were determined. SETTING: University of Texas Southwestern Medical Center, Dallas, Texas. PARTICIPANTS: Physicians enrolled in the University of Texas Southwestern Medical Center Obstetrics and Gynecology residency training program. RESULTS: Seventy-one of 73 residents participated. Knots tied with 0-caliber sutures had a higher mean failure load than those tied with 3-0 caliber sutures. For each type and caliber of suture, there were no differences in failure load between each level of residency training. However, senior residents tied knots with shorter loop lengths and had a lower proportion of knots that unraveled or slipped. CONCLUSIONS: Even though there were no differences in failure loads, senior residents tied tighter and more secure knots than their junior counterparts.

Heptanoate as a neural fuel: energetic and neurotransmitter precursors in normal and glucose transporter I-deficient (G1D) brain.

It has been postulated that triheptanoin can ameliorate seizures by supplying the tricarboxylic acid cycle with both acetyl-CoA for energy production and propionyl-CoA to replenish cycle intermediates. These potential effects may also be important in other disorders associated with impaired glucose metabolism because glucose supplies, in addition to acetyl-CoA, pyruvate, which fulfills biosynthetic demands via carboxylation. In patients with glucose transporter type I deficiency (G1D), ketogenic diet fat (a source only of acetyl-CoA) reduces seizures, but other symptoms persist, providing the motivation for studying heptanoate metabolism. In this work, metabolism of infused [5,6,7-(13)C(3)]heptanoate was examined in the normal mouse brain and in G1D by (13)C-nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). In both groups, plasma glucose was enriched in (13)C, confirming gluconeogenesis from heptanoate. Acetyl-CoA and glutamine levels became significantly higher in the brain of G1D mice relative to normal mice. In addition, brain glutamine concentration and (13)C enrichment were also greater when compared with glutamate in both animal groups, suggesting that heptanoate and/or C5 ketones are primarily metabolized by glia. These results enlighten the mechanism of heptanoate metabolism in the normal and glucose-deficient brain and encourage further studies to elucidate its potential antiepileptic effects in disorders of energy metabolism.

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet (13)C NMR of the adult mouse brain.

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the (13)C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-(13)C(2)]glucose (oxidized by both neurons and glia) and [1,2-(13)C(2)]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by (13)C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of (13)C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The (13)C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-(13)C]acetate relative to [1,6-(13)C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron-glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.

Glut1 deficiency (G1D): epilepsy and metabolic dysfunction in a mouse model of the most common human phenotype.

Brain glucose supplies most of the carbon required for acetyl-coenzyme A (acetyl-CoA) generation (an important step for myelin synthesis) and for neurotransmitter production via further metabolism of acetyl-CoA in the tricarboxylic acid (TCA) cycle. However, it is not known whether reduced brain glucose transporter type I (GLUT-1) activity, the hallmark of the GLUT-1 deficiency (G1D) syndrome, leads to acetyl-CoA, TCA or neurotransmitter depletion. This question is relevant because, in its most common form in man, G1D is associated with cerebral hypomyelination (manifested as microcephaly) and epilepsy, suggestive of acetyl-CoA depletion and neurotransmitter dysfunction, respectively. Yet, brain metabolism in G1D remains underexplored both theoretically and experimentally, partly because computational models of limited brain glucose transport are subordinate to metabolic assumptions and partly because current hemizygous G1D mouse models manifest a mild phenotype not easily amenable to investigation. In contrast, adult antisense G1D mice replicate the human phenotype of spontaneous epilepsy associated with robust thalamocortical electrical oscillations. Additionally, and in consonance with human metabolic imaging observations, thalamus and cerebral cortex display the lowest GLUT-1 expression and glucose uptake in the mutant mouse. This depletion of brain glucose is associated with diminished plasma fatty acids and elevated ketone body levels, and with decreased brain acetyl-CoA and fatty acid contents, consistent with brain ketone body consumption and with stimulation of brain beta-oxidation and/or diminished cerebral lipid synthesis. In contrast with other epilepsies, astrocyte glutamine synthetase expression, cerebral TCA cycle intermediates, amino acid and amine neurotransmitter contents are also intact in G1D. The data suggest that the TCA cycle is preserved in G1D because reduced glycolysis and acetyl-CoA formation can be balanced by enhanced ketone body utilization. These results are incompatible with global cerebral energy failure or with neurotransmitter depletion as responsible for epilepsy in G1D and point to an unknown mechanism by which glycolysis critically regulates cortical excitability.

Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors.

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-(13) C(2) ]glucose. The [3-(13) C]lactate/[2,3-(13) C(2) ]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, (13) C-(13) C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, (13) C multiplets of γ-aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that (13) C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.

High-resolution detection of ¹³C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy.

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization. The rate of brain glucose metabolism can be computed using (13)C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in (13)C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent (13)C carbons (multiplets) can provide additional information to (13)C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-(13)C(2)]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this infusion protocol, the (13)C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders.

Nonlinear dynamics of seizure prediction in a rodent model of epilepsy.

Epilepsy is a dynamical disorder with intermittent crises (seizures) that until recently were considered unpredictable. In this study, we investigated the predictability of epileptic seizures in chronically epileptic rats as a first step towards a subsequent timely intervention for seizure control. We look at the epileptic brain as a nonlinear complex system that undergoes spatio-temporal state transitions and the Lyapunov exponents as indices of its stability. We estimated the spatial synchronization or desynchronization of the maximum short-term Lyapunov exponents (STLmax, approximate measures of chaos) among multiple brain sites over days of electroencephalographic (EEG) recordings from 5 rats that had developed chronic epilepsy according to the lithium pilocarpine rodent model of epilepsy. We utilized this synchronization of EEG dynamics for the construction of a robust seizure prediction algorithm. The parameters of the algorithm were optimized using receiver operator curves (ROCs) on training EEG datasets from each rat for the algorithm to provide maximum sensitivity and specificity in the prediction of their seizures. The performance of the algorithm was then tested on long-term testing EEG datasets per rat. The thus optimized prediction algorithm on the testing datasets over all rats yielded a seizure prediction mean sensitivity of 85.9%, specificity of 0.180 false predictions per hour, and prediction time of 67.6 minutes prior to a seizure onset. This study provides evidence that prediction of seizures is feasible through analysis of the EEG within the framework of nonlinear dynamics, and thus paves the way for just-in-time pharmacological or physiological inter-ventions to abort seizures tens of minutes before their occurrence.

Control of synchronization of brain dynamics leads to control of epileptic seizures in rodents.

We have designed and implemented an automated, just-in-time stimulation, seizure control method using a seizure prediction method from nonlinear dynamics coupled with deep brain stimulation in the centromedial thalamic nuclei in epileptic rats. A comparison to periodic stimulation, with identical stimulation parameters, was also performed. The two schemes were compared in terms of their efficacy in control of seizures, as well as their effect on synchronization of brain dynamics. The automated just-in-time (JIT) stimulation showed reduction of seizure frequency and duration in 5 of the 6 rats, with significant reduction of seizure frequency (>50%) in 33% of the rats. This constituted a significant improvement over the efficacy of the periodic control scheme in the same animals. Actually, periodic stimulation showed an increase of seizure frequency in 50% of the rats, reduction of seizure frequency in 3 rats and significant reduction in 1 rat. Importantly, successful seizure control was highly correlated with desynchronization of brain dynamics. This study provides initial evidence for the use of closed-loop feedback control systems in epileptic seizures combining methods from seizure prediction and deep brain stimulation.

Information flow and application to epileptogenic focus localization from intracranial EEG.

Transfer entropy ( TE) is a recently proposed measure of the information flow between coupled linear or nonlinear systems. In this study, we suggest improvements in the selection of parameters for the estimation of TE that significantly enhance its accuracy and robustness in identifying the direction and the level of information flow between observed data series generated by coupled complex systems. We show the application of the improved TE method to long (in the order of days; approximately a total of 600 h across all patients), continuous, intracranial electroencephalograms (EEG) recorded in two different medical centers from four patients with focal temporal lobe epilepsy (TLE) for localization of their foci. All patients underwent ablative surgery of their clinically assessed foci. Based on a surrogate statistical analysis of the TE results, it is shown that the identified potential focal sites through the suggested analysis were in agreement with the clinically assessed sites of the epileptogenic focus in all patients analyzed. It is noteworthy that the analysis was conducted on the available whole-duration multielectrode EEG, that is, without any subjective prior selection of EEG segments or electrodes for analysis. The above, in conjunction with the use of surrogate data, make the results of this analysis robust. These findings suggest a critical role TE may play in epilepsy research in general, and as a tool for robust localization of the epileptogenic focus/foci in patients with focal epilepsy in particular.