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Brain biospecimen banking requires centralized resources, national networks for referral of donors, trained personnel to interact with grieving families, and scientific staff to process the biospecimens. Process development of quality control standards is needed to meet the specific requirements of emerging genomic and proteomic technologies. Attention has to be paid to agonal factors and postmortem interval, tissue processing, neuropathology review, and long-term storage. Samples of both diseased and unaffected normal tissues are required with age- and gender-matched control tissues. Data management is vital to store and retrieve quality control measures, clinical and pathologic data linked to the biospecimens. Customized solutions for managing the acquisition and long-term storage of high-quality brain and tissue biospecimens is necessary to support neuroscience research programs, biomarker discovery and genome scale technologies. Biorepositories that operate according to best-practice policies and procedures guarantee the final wish of the families who donate tissue to support neuroscience research and discovery science.

Brain biospecimen banking requires centralized resources, national networks for referral of donors, trained personnel to interact with grieving families, and scientific staff to process the biospecimens. Process development of quality control standards is needed to meet the specific requirements of emerging genomic and proteomic technologies. Attention has to be paid to agonal factors and postmortem interval, tissue processing, neuropathology review, and long-term storage. Samples of both diseased and unaffected normal tissues are required with age- and gender-matched control tissues. Data management is vital to store and retrieve quality control measures, clinical and pathologic data linked to the biospecimens. Customized solutions for managing the acquisition and long-term storage of high-quality brain and tissue biospecimens is necessary to support neuroscience research programs, biomarker discovery and genome scale technologies. Biorepositories that operate according to best-practice policies and procedures guarantee the final wish of the families who donate tissue to support neuroscience research and discovery science.

Contains fulltext : 165723pub.pdf (Publisher’s version ) (Closed access) Intracranial volume reflects the maximally attained brain size during development, and remains stable with loss of tissue in late life. It is highly heritable, but the underlying genes remain largely undetermined. In a genome-wide association study of 32,438 adults, we discovered five previously unknown loci for intracranial volume and confirmed two known signals. Four of the loci were also associated with adult human stature, but these remained associated with intracranial volume after adjusting for height. We found a high genetic correlation with child head circumference (rhogenetic = 0.748), which indicates a similar genetic background and allowed us to identify four additional loci through meta-analysis (Ncombined = 37,345). Variants for intracranial volume were also related to childhood and adult cognitive function, and Parkinson's disease, and were enriched near genes involved in growth pathways, including PI3K-AKT signaling. These findings identify the biological underpinnings of intracranial volume and their link to physiological and pathological traits.

Epilepsy is a common chronic neurological disorder characterized by seizures. Mortality is significantly increased in patients with epilepsy. Sudden unexpected death in epilepsy (SUDEP) is the most common seizure-related category of death. A retrospective study of forensic autopsy cases from 2007 to 2009 at the Office of the Chief Medical Examiner (OCME) yielded a total of 104 sudden unexpected deaths directly or indirectly caused by an epilepsy/seizure disorder in the State of Maryland. Of these deaths, 74 cases met a general accepted definition of SUDEP. The age of SUDEP individuals ranged from 14 to 63 with the majority of subjects in the ages between 21 and 50 years (58 cases, 78.4%). Males were slightly more likely than females to die of SUDEP (male:female=1.5:1 based on the rate). The onset age of epilepsy was documented in 47.3% of cases (35/74) based on investigation and medical records. Of the 35 cases, 12 subjects had early onset epilepsy (onset ages 1-15 years) and 20 subjects had duration of epilepsy for more than 10 years. The majority of deaths (61 of the 74 cases, 82.4%) were unwitnessed. Death scene investigation showed that 71 deaths (95.9%) occurred inside their residence with 50 subjects (70.4%) found either in bed or on the bedroom floor near the bed. Forty-three out of 74 cases (58.1%) showed neuropathological lesions. Per history, 50 subjects were reported as being on anti-epileptic drugs (AEDs). However, postmortem toxicological analysis revealed that only 26 subjects (35.1%) had detectable AEDs. Of the 74 cases, seizure disorder or epilepsy was listed as primary cause of death in 66 cases and the term of SUDEP as official cause of death in only 8 cases. This report focuses on the characteristics of death scene investigation and postmortem examination findings of SUDEP cases.

Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ~900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography— the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.

The Office of the Chief Medical Examiner of Maryland recorded a total of 149 drug abuse deaths of teenagers aged 13–19 years between 1991 and 2006. Of these deaths, 96 (64.4%) were caused by the use of narcotic drugs only, 29 (19.5%) by both narcotics and cocaine, four (2.7%) by both narcotics and methylenedioxymethamphetamine, six (4.0%) by cocaine only, and 14 (9.4%) by volatile substances (e.g., butane, Freon, nitrous oxide, and propane). The annual death rate from drug abuse for teenagers increased from 1.4 deaths per 100,000 population in 1991 to 2.7 deaths per 100,000 population in 2006 (chi-square test for time trend, p

It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l-[U-14C]glutamate, label incorporation into [14C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l-[U-14C]glutamate into [14C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.

Although the majority of agents with antiexcitotoxic action act as glutamate receptor antagonists, enzymatic degradation of glutamate can also be neuroprotective. The very low specific activity of the mammalian form of glutamate decarboxylase (GAD), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA) from glutamate in neurons, is likely to limit its utility as an antiglutamate neuroprotectant. In contrast, the bacterial form of GAD can be isolated with relatively high specific activity and is most active in acidic environments. We have expressed and purified GAD from Escherichia coli (bGAD) and tested the ability of the enzyme to protect against glutamate excitotoxicity. Incubation of rat hipppocampal slices with the potassium channel antagonist tetraethyl ammonium (TEA) resulted in widespread excitotoxic death of pyramidal and granule cell neurons. bGAD alone showed no significant neurotoxicity and significantly reduced excitotoxicity induced by TEA. We hypothesize that bGAD may be internalized into the synaptic vesicle compartment by nonspecific endocytosis, where both the appropriate pH and high glutamate concentrations are present. Targeting of this enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant against excitotoxicity.

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 μmol/L fluorocitrate. However, 20 and 100 μmol/L fluorocitrate reduced lactate oxidation by 55 ± 20% and 68 ± 12%, respectively (p

The oxidative capacity of the brain for alternate substrates, glucose, lactate, pyruvate, acetate, glutamate, and glutamine was determined by using microdialysis to infuse 14C-labeled compounds into the interstitial fluid of adult rat brain and by collecting the brain-generated 14CO2 from the dialysis eluate. All compounds were readily oxidized. The recovery of 14CO2 was enhanced for those compounds metabolically close to entry into the TCA cycle or known to have a low interstitial concentration. Two compounds, pyruvate and lactate, demonstrated reciprocal competition when added as nonradioactive competitors. Oxidation of two amino acids, 14C-glutamate and 14C-glutamine, was stimulated by the addition of nonradioactive acetate and pyruvate. α-Cyano-4-hydroxycinnamate decreased 14C-lactate and 14C-pyruvate oxidation, consistent with the transport of both compounds via a monocarboxylate transporter. The results of this in vivo study support the results of previous in vitro studies that showed that a wide range of compounds formed from glucose in the brain are also oxidized in the brain for energy production. © 2007 Wiley-Liss, Inc.

Solid tissues are critical for child-health research. Specimens are commonly obtained at the time of biopsy/surgery or postmortem. Research tissues can also be obtained at the time of organ retrieval for donation or from tissue that would otherwise have been discarded. Navigating the ethics of solid tissue collection from children is challenging, and optimal handling practices are imperative to maximize tissue quality. Fresh biopsy/surgical specimens can be affected by a variety of factors, including age, gender, BMI, relative humidity, freeze/thaw steps, and tissue fixation solutions. Postmortem tissues are also vulnerable to agonal factors, body storage temperature, and postmortem intervals. Nonoptimal tissue handling practices result in nucleotide degradation, decreased protein stability, artificial posttranslational protein modifications, and altered lipid concentrations. Tissue pH and tryptophan levels are 2 methods to judge the quality of solid tissue collected for research purposes; however, the RNA integrity number, together with analyses of housekeeping genes, is the new standard. A comprehensive clinical data set accompanying all tissue samples is imperative. In this review, we examined: the ethical standards relating to solid tissue procurement from children; potential sources of solid tissues; optimal practices for solid tissue processing, handling, and storage; and reliable markers of solid tissue quality.

We studied how carbon monoxide (CO) is distributed within the human body through quantitation of CO concentrations in postmortem tissue samples from fatalities including possible CO exposure. Stored, frozen tissues were diced, sonicated in water, and 0.01-8.0 mg wet weight (ww) tissues were incubated with sulfosalicylic acid in CO-purged, septum-sealed vials. CO released into the headspace was quantitated by reduction gas chromatography. Mean tissue CO concentrations (pmol/mg ww) from subjects diagnosed to have no known CO exposure (control, N=14), died from fire (N=13), and CO asphyxiation (N=7), respectively, were: adipose (2;13;9), brain (3;13;65), muscle (15;97;297), heart (30;99;371), kidney (22;432;709, lung (54;690;2638), spleen (73;1366;3548), and blood (162;2238;5070). Carboxyhemoglobin concentrations were 1.4%, 25.2%, and 69.1% of total hemoglobin, respectively. We conclude that measurements of CO concentration in a variety of tissues can be used as markers for the degree of exogenous CO exposure and the identification of possible causes of death. Language: en

Several lines of work have shown that astrocytes release glutamate in response to receptor activation, which results in a modulation of local synaptic activity. Astrocytic glutamate release is Ca 2+ -dependent and occurs in conjunction with exocytosis of glutamate containing vesicles. However, astrocytes contain a millimolar concentration of cytosolic glutamate and express channels permeable to small anions, such as glutamate. Here, we tested the idea that astrocytes respond to receptor stimulation by dynamic changes in cell volume, resulting in volume-sensitive channel activation, and efflux of cytosolic glutamate. Confocal imaging and whole-cell recordings demonstrated that astrocytes exhibited a transient Ca 2+ -dependent cell volume increase, which activated glutamate permeable channels. HPLC analysis revealed that glutamate was released in conjunction with other amino acid osmolytes. Our observations indicate that volume-sensitive channel may constitute a previously uncharacterized target for modulation of astrocyte-neuronal interactions.

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

To evaluate the response of astrocytes in the auditory pathway to increased neuronal signaling elicited by acoustic stimulation, conscious rats were presented with a unilateral broadband click stimulus and functional activation was assessed by quantitative autoradiography using three tracers to pulse label different metabolic pools in brain: [2-14C]acetate labels the 'small' (astrocytic) glutamate pool, [1-14C]hydroxybutyrate labels the 'large' glutamate pool, and [14C]deoxyglucose, reflects overall glucose utilization (CMR(glc)) in all brain cells. CMR(glc) rose during brain activation, and increased activity of the oxidative pathway in working astrocytes during acoustic stimulation was registered with [2-14C]acetate. In contrast, the stimulation-induced increase in metabolic activity was not reflected by greater trapping of products of [1-14C]hydroxybutyrate. The [2-14C]acetate uptake coefficient in the inferior colliculus and lateral lemniscus during acoustic stimulation was 15% and 18% (p0.01) higher in the activated compared to contralateral hemisphere, whereas CMR(glc) in these structures rose by 66% (p0.01) and 42% (p0.05), respectively. Calculated rates of brain utilization of blood-borne acetate (CMR(acetate)) are about 15-25% of total CMR(glc) in non-stimulated tissue and 10-20% of CMR(glc) in acoustically activated structures; they range from 28 to 115% of estimated rates of glucose oxidation in astrocytes. The rise in acetate utilization during acoustic stimulation is modest compared to total CMR(glc), but astrocytic oxidative metabolism of 'minor' substrates present in blood can make a significant contribution to the overall energetics of astrocytes and astrocyte-neuron interactions in working brain.

Down syndrome (DS) results from complete or partial triplication of human chromosome 21. It is assumed that the neurological and other symptoms are caused by the overexpression of genes on chromosome 21, but this hypothesis has not yet been assessed on a chromosome-wide basis. Here we show that expression of genes localized to chromosome 21 is globally up-regulated in human fetal trisomy 21 cases, both in cerebral cortex extracts and in astrocytic cell lines cultured from cerebral cortex. This abnormal regulation of gene expression is specific to chromosome 21. Our data describe transcriptional changes that are specific to many genes assigned to chromosome 21 and do not directly measure the clinical phenotype of DS. However, it is possible that these gene expression changes ultimately relate to the phenotypic variability of DS.

Objectives. Recurrent hypoxemia has been proposed as an important pathophysiological mechanism underlying sudden infant death syndrome (SIDS). However, conflicting results emerged when xanthines were used as markers for hypoxia. The vascular endothelial growth factor (VEGF) gene is highly sensitive to changes in tissue partial oxygen tension, and changes in genomic and protein expression occur even after changes in oxygenation within the physiologic range.Methods. For determining whether hypoxia precedes SIDS, VEGF levels were measured using an enzyme-linked immunosorbent assay in the cerebrospinal fluid (CSF) of 51 SIDS infants and in 33 additional control infants who died of an identifiable cause. In addition, 6 rats that had a chronically implanted catheter in the lateral ventricle were exposed to a short hypoxic challenge, and VEGF concentrations were measured in CSF at various time points for 24 hours. Another set of 6 rats were killed with a pentobarbital overdose, and VEGF CSF levels were obtained at different time points after death.Results. Mean VEGF concentrations in CSF were 308.2 ± 299.1 pg/dL in the SIDS group and 85.1 ± 82.9 pg/dL in those who died of known causes. Mean postmortem delay averaged 22 hours for both groups. In rat experiments, hypoxic exposures induced time-dependent increases in VEGF, peaking at 12 hours and returning to baseline at 24 hours. Postmortem duration in the animals was associated with gradual increases in VEGF that reached significance only at 36 hours.Conclusions. We conclude that VEGF CSF concentrations are significantly higher in infants who die of SIDS. We postulate that hypoxia is a frequent event that precedes the sudden and unexpected death of these infants.

The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine ; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U- 13 C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U- 13 C]glutamate 85% of the 13 C metabolized was converted to glutamine. The formation of [1,2,3- 13 C 3 ]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2-0.5 mM glutamate, 13 C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [ 13 C]lactate was essentially unchanged within the range of 0.2-0.5 mM glutamate, whereas the amount of [ 13 C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.

Hydrolysis of extracellular glutamine as a potential source of increased extracellular glutamate in the quinolinic acid (QUIN)-injured brain of the unanesthetized, free-moving rat was examined by microdialysis and HPLC analysis. Injury was initiated by injection of 100 nmoles of QUIN into the hippocampus. Immediately postinjury or 24 hr postinjury, the injection site was perfused with artificial cerebrospinal fluid + (14)C-glutamine to measure its conversion to (14)C-glutamate. L-trans-pyrrolidine-2,4-dicarboxylate (L-PDC), a glutamate uptake inhibitor, was added to the perfusate to enhance the detection of extracellular (14)C-glutamate. QUIN injury was followed by an immediate increase in extracellular glutamate that persisted 24 hr later. When (14)C-glutamine was added to the perfusate, a significant amount of (14)C-glutamate was recovered, and it was greater following QUIN injury than in control animals (P < 0.001). Up to 32% of the extracellular (14)C-glutamine was converted to (14)C-glutamate following QUIN injury. Considering the high concentration of glutamine normally present in the extracellular fluid, glutamine hydrolysis is a potential and important source for the increase in extracellular glutamate after neuronal injury in vivo.

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

The rate of glutaminase-dependent metabolism of glutamine in intact astrocytes was determined under conditions in which the extracellular concentration of glutamine was varied between 0.2 and 3.2 mM glutamine for control and dibutyryl cyclic AMP (dBcAMP)-treated cells. Glutamine metabolism by intact cells increased with increasing extracellular glutamine when calculations were based on the extracellular specific activity of glutamine. However, when the rate was based on the intracellular specific activity of glutamine, the rate of glutamine metabolism was independent of the media glutamine concentration. Similar results were obtained when cells were treated with dBcAMP, although the rates were approximately twice as high compared to untreated cells. The rate of formation of 14CO2 from [1-14C]glutamine and [1-14C]glutamate, based on the extracellular specific activities, were 93 +/- 5 and 40 +/- 4 nmol/mg protein/h, respectively. Oxidation rates based on the experimentally determined intracellular specific activity of glutamine and glutamate were 144 +/- 8 and 209 +/- 18 nmol/mg protein/h, respectively. In dBcAMP-treated astrocytes, the oxidation rates were higher than in untreated cells. These studies demonstrate that determination of the specific activity of compounds inside the cell aids in the interpretation of metabolic studies with intact cells and that both the initial steps of glutamine metabolism and the rate of 14CO2 formation from 14C-glutamine via the TCA cycle were increased in dBcAMP-treated astrocytes.

Detailed anatomical understanding of the human brain is essential for unraveling its functional architecture, yet current reference atlases have major limitations such as lack of whole‐brain coverage, relatively low image resolution, and sparse structural annotation. We present the first digital human brain atlas to incorporate neuroimaging, high‐resolution histology, and chemoarchitecture across a complete adult female brain, consisting of magnetic resonance imaging (MRI), diffusion‐weighted imaging (DWI), and 1,356 large‐format cellular resolution (1 µm/pixel) Nissl and immunohistochemistry anatomical plates. The atlas is comprehensively annotated for 862 structures, including 117 white matter tracts and several novel cyto‐ and chemoarchitecturally defined structures, and these annotations were transferred onto the matching MRI dataset. Neocortical delineations were done for sulci, gyri, and modified Brodmann areas to link macroscopic anatomical and microscopic cytoarchitectural parcellations. Correlated neuroimaging and histological structural delineation allowed fine feature identification in MRI data and subsequent structural identification in MRI data from other brains. This interactive online digital atlas is integrated with existing Allen Institute for Brain Science gene expression atlases and is publicly accessible as a resource for the neuroscience community. J. Comp. Neurol. 524:3127–3481, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

The Office of the Chief Medical Examiner of Maryland recorded a total of 149 drug abuse deaths of teenagers aged 13-19 years between 1991 and 2006. Of these deaths, 96 (64.4%) were caused by the use of narcotic drugs only, 29 (19.5%) by both narcotics and cocaine, four (2.7%) by both narcotics and methylenedioxymethamphetamine, six (4.0%) by cocaine only, and 14 (9.4%) by volatile substances (e.g., butane, Freon, nitrous oxide, and propane). The annual death rate from drug abuse for teenagers increased from 1.4 deaths per 100,000 population in 1991 to 2.7 deaths per 100,000 population in 2006 (chi-square test for time trend, p0.01). The increase in teenager drug abuse deaths occurred in 1999 and since has remained at a higher rate. Further analysis revealed that the increase in drug abuse deaths was attributable to a large degree to narcotic drugs, particularly heroin/morphine and methadone, and was confined to teenagers residing in the suburban and rural areas.

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce 14C-labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to 14CO2. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO2. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.

Morphological plasticity in response to estradiol is a hallmark of astrocytes in the arcuate nucleus. The functional consequences of these morphological changes have remained relatively unexplored. Here we report that in the arcuate nucleus estradiol significantly increased the protein levels of the two enzymes in the glutamate-glutamine cycle, glutamine synthetase and glutaminase. We further demonstrate that these estradiol-mediated changes in the enzyme protein levels may underlie functional changes in neurotransmitter availability as: 1) total glutamate concentration in the arcuate nucleus was significantly increased and 2) microdialysis revealed a significant increase in extracellular glutamate levels after a synaptic challenge in the presence of estradiol. These data implicate the glutamate-glutamine cycle in the generation and/or maintenance of glutamate and suggest that the difference in extracellular glutamate between estradiol- and oil-treated animals may be related to an increased efficiency of the cycle enzymes. In vivo enzyme activity assays revealed that the estradiol mediated increase in glutamate-glutamine cycle enzymes in the arcuate nucleus led to an increase in gamma-aminobutyric acid and is likely not related to the increase in extracellular glutamate. Thus, we have observed two-independent effects of estradiol on amino acid neurotransmission in the arcuate nucleus. These data suggest a possible functional consequence of the well-established changes in glial morphology that occur in the arcuate nucleus in the presence of estradiol and suggest the importance of neuronal-glial cooperation in the regulation of hypothalamic functions such as food intake and body weight.

Gene expression profiles were assessed in the hippocampus, entorhinal cortex, superior-frontal gyrus, and postcentral gyrus across the lifespan of 55 cognitively intact individuals aged 20–99 years. Perspectives on global gene changes that are associated with brain aging emerged, revealing two overarching concepts. First, different regions of the forebrain exhibited substantially different gene profile changes with age. For example, comparing equally powered groups, 5,029 probe sets were significantly altered with age in the superior-frontal gyrus, compared with 1,110 in the entorhinal cortex. Prominent change occurred in the sixth to seventh decades across cortical regions, suggesting that this period is a critical transition point in brain aging, particularly in males. Second, clear gender differences in brain aging were evident, suggesting that the brain undergoes sexually dimorphic changes in gene expression not only in development but also in later life. Globally across all brain regions, males showed more gene change than females. Further, Gene Ontology analysis revealed that different categories of genes were predominantly affected in males vs. females. Notably, the male brain was characterized by global decreased catabolic and anabolic capacity with aging, with down-regulated genes heavily enriched in energy production and protein synthesis/transport categories. Increased immune activation was a prominent feature of aging in both sexes, with proportionally greater activation in the female brain. These data open opportunities to explore age-dependent changes in gene expression that set the balance between neurodegeneration and compensatory mechanisms in the brain and suggest that this balance is set differently in males and females, an intriguing idea.

A 4.6-fold increase in interstitial glutamate was observed following the reverse microdialysis of 5 mM glutamine into the rat hippocampus. Two possible mechanisms of glutamine hydrolysis were examined: (a) an enzymatic glutaminase activity and (b) a non-enzymatic mechanism. Injection of 14C-glutamine at the site of microdialysis followed by microdialysis with artificial cerebral spinal fluid allowed isolation of 14C-glutamine (63%), 14C-glutamate (14%), and a compound tentatively identified as pyroglutamate (22%). In this study, we determined if non-enzymatic pyroglutamate formation from glutamine contributed to the synthesis of glutamate. Pyroglutamate is in chemical equilibrium with glutamate, although under physiological conditions, the chemical equilibrium is strongly in the direction of pyroglutamate. In vitro stability studies indicated that 14C-glutamine and 14C-pyroglutamate are not subject to significant non-enzymatic breakdown at pH 6.5-7.5 at 37 degrees C for up to 8 h. Reverse microdialysis with 1 mM pyroglutamate did not increase interstitial glutamate levels. Following injection of 14C-pyroglutamate and microdialysis, radioactivity was recovered in 14C-pyroglutamate (88%) and 14C-glutamine (11%). Less than 1% of the radioactivity was recovered as glutamate. Our data do not support a role of pyroglutamate as an intermediate in the formation of extracellular glutamate following the infusion of glutamine. However, it confirms that pyroglutamate, a known constituent in brain, is actively metabolized in brain cells and contributes to glutamine in the interstitial space.

It is a major recent finding that astrocytes can influence synaptic activity by release of glutamate, but many other glutamate-mediated activities are also controlled by astrocytes. Even the most obvious neuronal function of glutamate - its release as a transmitter - is regulated by astrocytes; these cells are needed for formation of precursors for glutamate synthesis, for reuptake of released transmitter, and for disposal of excess glutamate. Without astrocytic involvement, normal function of glutamatergic neurons is not possible, as exemplified by almost instantaneous abrogation of normal vision and learning upon inhibition of astrocyte-specific metabolic pathways. In addition, astrocytes are essential for production of the neuroprotectant glutathione, yet they can also contribute to neuronal death during ischemia by maintaining glutamine synthesis, enabling neuronal formation of neurotoxic glutamate.

Carboxyhemoglobin (COHb) levels were determined in stored blood samples from 91 infants diagnosed to have died from the sudden infant death syndrome (SIDS) (0.59+/-0.41%, excluding one outlying value of 10.83%); 48 age-matched controls (0.53+/-0.38%); and three individuals who died from fire related causes (41+/-20%). No statistical differences in COHb levels were detected between blood from SIDS and control infants (p = 0.43).

Maple syrup urine disease (MSUD) and phenylketonuria (PKU) are associated with accumulation of large neutral amino acids (LNAA) in blood and tissues and a decrease of other LNAA not directly related to the enzyme defects. One characteristic shared by both the elevated and decreased amino acids is that all are substrates for transport via the large neutral amino acid transporter. In this study, the blood brain barrier was effectively bypassed using microdialysis to determine the immediate effect of infused phenylalanine, tyrosine, 2-amino-2-norborane-carboxylic acid (BCH), and leucine and alpha-ketoisocaproate on extracellular levels of LNAA. The concentration of non-infused LNAA increased in the interstitial fluid, presumably due to trans-stimulated exchange of these LNAA from intracellular pools as the infused LNAA entered the cells. Such trans-stimulated exchange can potentially deplete cells of multiple essential LNAA. It is proposed that brain cells in disorders such as MSUD and PKU may be subject to two mechanisms that limit the availability of a full complement of these amino acids: competition for transport of LNAAs at the blood brain barrier and trans-stimulated exchange out of neuronal cells for subsequent metabolism or sequestration in the periphery.

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

The present study determined the metabolic fate of [U-13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mM alpha-ketoisocaproate (alpha-KIC). When astrocytes were incubated with 0.2 mM [U-13C]glutamate, 64.1% of the 13C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-(13)C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of alpha-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3-(13)C3-isotopomer of lactate in cells incubated in the presence of alpha-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of alpha-KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of alpha-KIC than in control cells. Overall, the results demonstrate that the presence of alpha-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.

An assay for the determination of l -glutaminase in extracts and intact cells is described. The method is based on the stereospecific release of 3 H 2 O from l -[2- 3 H]glutamine when l -glutaminase is coupled to l -aspartate:2-oxoglutarate amino transferase. The substrate, glutamine, and intermediate product, glutamate, are separated from the final reaction product, tritiated water, on a mixed-bed ion-exchange column which retains amino acids and organic acids but not water. The method has been adapted to determine the activity of l -glutaminase in cultured human diploid fibroblasts.

Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.

Incorporation of [14C] from acetoacetate, D(-)- and L(+)-3-hydroxybutyrate, glucose, glutamine, acetate and palmitate in cellular lipids were studied in cultures in human diploid fibroblasts (HDF). The results showed that acetoacetate was 2-10 times more effective as a lipogenic precursor than was either D- or L-3-hydroxybutyrate. Its extent of incorporation into lipids was 2- to 8-fold more than the other precursors examined under conditions when the overall rates of nonsaponifiable and saponifiable lipogenesis as measured by 3H2O incorporation were essentially unchanged. Acetoacetate supported both saponifiable and nonsaponifiable lipid synthesis with half-saturation values (Km app.) of 185 microM and 30 microM, respectively. Glucose stimulated acetoacetate incorporation into lipids whereas, conversely, acetoacetate inhibited [14C]glucose incorporation into lipids. The presence of low density lipoproteins (LDL) cholesterol (40 microgram cholesterol/mL) inhibited the incorporation of [14C] from acetoacetate 56% into nonsaponifiable lipids; the inhibition was consistently higher (75%) when [14C]glucose or glutamine were the precursors. The loss of 3-hydroxy-3-methyl-glutaryl CoA (HMG CoA) reductase activity upon addition of LDL-cholesterol was greater than the suppression of [14C] incorporation from acetoacetate or glucose into nonsaponifiable lipids. In the presence of glucose, [14C]acetoacetate was incorporated into 3-beta OH sterols (digitonin precipitable) 7.7 +/- 1.1 times more effectively than was [14C]glucose. The results suggest that HDF would be a suitable model to investigate the effects of various precursors of HMG CoA on the rate of cholesterol biosynthesis.

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts was studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, d -3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.

Synthesis and turnover of nitrate reductase molecules and soluble proteins of tobacco XD cells were studied by labeling pre-existing proteins with 14C-arginine to make them radioactive and with 99 atom % 15N to increase their buoyant density. The cells were then transferred to medium in which new proteins were synthesized from 14N and were radioactively labeled with 3H-arginine. The breakdown of 15N-labeled proteins was followed after transfer of cells to 14N medium by means of the 14C label. The influence of the pre-existing pool of 15N-amino acids on the density of newly synthesized proteins was followed by means of the 3H label. At various times protein was extracted and subjected to isopycnic equilibrium centrifugation in CsCl. Buoyant densities, relative to an added standard, catalase, were determined for nitrate reductase activity, 14C-labeled protein, and 3H-labeled protein. The studies were carried out (a) during induction of nitrate reductase activity, (b) after full induction, when the level of nitrate reductase activity remains constant, and (c) after a shift from inducing to noninducing conditions, when nitrate reductase activity decays. The buoyant density data show that nitrate reductase activity induced by nitrate is an activity of a protein synthesized de novo after addition of inducer. However, even when the enzyme level was constant, the buoyant density of nitrate reductase decreased from that of 15N-nitrate reductase towards that of 14N-nitrate reductase, indicating that the enzyme turned over. Furthermore, nitrate reductase molecules continued to be synthesized as nitrate reductase activity decayed under noninducing conditions. Turnover of both pre-existing proteins labeled with 14C and newly synthesized proteins labeled with 3H was also observed.

The relative contributions of radioactively labeled fatty acids and glucose to the synthesis of glutamate and glutamine were compared in native and dibutyryl cyclic AMP (diBcAMP)-treated primary rat astrocytes. The intracellular specific activities of glutamate and glutamine were 10-fold greater than the specific activities of aspartate or alanine. Butyrate, octanoate and palmitate were equally as effective as precursors for glutamate and glutamine while glucose was 50% as effective as the fatty acids. The specific activity of glutamate and glutamine were identical in the absence of diBcAMP. In diBcAMP treated cells the specific activity of glutamine was greater than that of glutamate when octanoate and palmitate were the labeled precursors. This suggests that cultured astrocytes preferentially utilize free fatty acids for glutamate/glutamine synthesis and that diBcAMP-treated astrocytes contain more than one glutamate compartment.

Short term administration of methylxanthines has been reported to alter levels and turnover rates of brain catecholamines and indoles. In the present study continuous administration of caffeine and theophylline was achieved by subcutaneous implantation of silastic tubing filled with powdered methylxanthines. Serum levels of caffeine and theophylline were monitored daily for 2 weeks by high performance liquid chromatography (HPLC) and averaged 35μM and 7μM, respectively. After 2 weeks of continuous exposure to methylxanthines the dopamine level and turnover rate were unaltered from control in the neostriatum, hypothalamus and cortex. Likewise the level and turnover of norepinephrine were unaltered from control in the cerebellum, hypothalamus and cortex. Also unaffected were the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA), serotonin and 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus and cortex. These results indicate that in mice the continuous exposure to methylxanthines has no long lasting effect on monoamine neurotransmitters in the brain.

The quantity of aspartate, but not of glutamate, synthesized by human diploid fibroblasts (HDF) was inversely proportional to the glucose concentration. Forty hours after confluent cells were refed with media containing 1.5 mM or 55 microM glucose, the aspartate concentration was 8 microM in medium high in glucose and 28 microM in medium low in glucose; glutamate was 350 microM at both glucose levels. The label incorporation from [U-14C] glutamine (0.8 C/ml) into aspartate after 40 hours in 1.5 mM glucose medium was less then 1000 DPM/ml and into glutamate 540,000 DPM/ml. The respective label incorporation into aspartate in 55 microM glucose medium was 118,000 DPM/ml and into glutamate 450,000 DPM/ml. In media with 1.5 mM glucose, label incorporation into both aspartate and glutamate was observed from [6-14C] glucose, [3-14C] pyruvate, and [1-14C] pyruvate. Since [1-14C] pyruvate labeled aspartate in 1.5 mM glucose medium, whereas [U-14C] glutamine did not, the observations support a cystolic pathway of aspartate synthesis from glycolytic intermediates in the presence of glucose. However, when the glucose concentration is decreased, glutamine appears to be the primary carbon source for aspartate synthesis. Therefore, both the quantity and the pathway of aspartate synthesis in HDF are functions of the glucose concentration. It is postulated that increased accumulation of aspartate in the media of HDF at confluency may be explained by decreased availability of acetyl CoA or of NADH.