Quantification of brain glycogen concentration and turnover through localized 13C NMR of both the C1 and C6 resonances.

We have recently shown that at isotopic steady state (13)C NMR can provide a direct measurement of glycogen concentration changes, but that the turnover of glycogen was not accessible with this protocol. The aim of the present study was to design, implement and apply a novel dual-tracer infusion protocol to simultaneously measure glycogen concentration and turnover. After reaching isotopic steady state for glycogen C1 using [1-(13)C] glucose administration, [1,6-(13)C(2)] glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected (13)C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, we implemented 2D gradient based localization using the Fourier series window approach, in conjunction with time-domain analysis of the resulting FIDs using jMRUI. The glycogen concentration of 5.1 +/- 1.6 mM measured from the C1 position was in excellent agreement with concomitant biochemical determinations. Glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the alpha-chloralose anesthetized rat was 0.7 micromol/g/h.

Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain.

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo- and exocytosis. Here we describe the identification and functional characterization of a novel H(+)-myo- inositol co-transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo-inositol and related stereoisomers with a Michaelis-Menten constant of approximately 100 microM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75-90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H(+)-coupled myo- inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo-inositol brain metabolism.

1H-[13C] NMR spectroscopy of the rat brain during infusion of [2-13C] acetate at 14.1 T.

Full signal intensity (1)H-[(13)C] NMR spectroscopy, combining a preceding (13)C-editing block based on an inversion BISEP (B(1)-insensitive spectral editing pulse) with a spin-echo coherence-based localization, was developed and implemented at 14.1 T. (13)C editing of the proposed scheme was achieved by turning on and off the (13)C adiabatic full passage in the (13)C-editing block to prepare inverted and noninverted (13)C-coupled (1)H coherences along the longitudinal axis prior to localization. The novel (1)H-[(13)C] NMR approach was applied in vivo under infusion of the glia-specific substrate [2-(13)C] acetate. Besides a approximately 50% improvement in sensitivity, spectral dispersion was enhanced at 14.1 T, especially for J-coupled metabolites such as glutamate and glutamine. A more distinct spectral structure at 1.9-2.2 ppm(parts per million) was observed, e.g., glutamate C3 showed a doublet pattern in both simulated (1)H spectrum and in vivo (13)C-edited (1)H NMR spectra. Besides (13)C time courses of glutamate C4 and glutamine C4, the time courses of glutamate C3 and glutamine C3 obtained by (1)H-[(13)C] NMR spectroscopy were reported for the first time. Such capability should greatly improve the ability to study neuron-glial metabolism using (1)H-observed (13)C-edited NMR spectroscopy.

Creatine and guanidinoacetate transport at blood-brain and blood-cerebrospinal fluid barriers.

While it was thought that most of cerebral creatine is of peripheral origin, AGAT and GAMT are well expressed in CNS where brain cells synthesize creatine. While the creatine transporter SLC6A8 is expressed by microcapillary endothelial cells (MCEC) at blood-brain barrier (BBB), it is absent from their surrounding astrocytes. This raised the concept that BBB has a limited permeability for peripheral creatine, and that the brain supplies a part of its creatine by endogenous synthesis. This review brings together the latest data on creatine and guanidinoacetate transport through BBB and blood-CSF barrier (BCSFB) with the clinical evidence of AGAT-, GAMT- and SLC6A8-deficient patients, in order to delineate a clearer view on the roles of BBB and BCSFB in the transport of creatine and guanidinoacetate between periphery and CNS, and on brain synthesis and transport of creatine. It shows that in physiological conditions, creatine is taken up by CNS from periphery through SLC6A8 at BBB, but in limited amounts, and that CNS also needs its own creatine synthesis. No uptake of guanidinoacetate from periphery occurs at BBB except under GAMT deficiency, but a net exit of guanidinoacetate seems to occur from CSF to blood at BCSFB, predominantly through the taurine transporter TauT.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.

Brain Surface Segmentation of Magnetic Resonance Images of the Fetus

In this work we present a method for the image analysisof Magnetic Resonance Imaging (MRI) of fetuses. Our goalis to segment the brain surface from multiple volumes(axial, coronal and sagittal acquisitions) of a fetus. Tothis end we propose a two-step approach: first, a FiniteGaussian Mixture Model (FGMM) will segment the image into3 classes: brain, non-brain and mixture voxels. Second, aMarkov Random Field scheme will be applied tore-distribute mixture voxels into either brain ornon-brain tissue. Our main contributions are an adaptedenergy computation and an extended neighborhood frommultiple volumes in the MRF step. Preliminary results onfour fetuses of different gestational ages will be shown.

Anemia and brain oxygen after severe traumatic brain injury.

PURPOSE: To investigate the relationship between hemoglobin (Hgb) and brain tissue oxygen tension (PbtO(2)) after severe traumatic brain injury (TBI) and to examine its impact on outcome. METHODS: This was a retrospective analysis of a prospective cohort of severe TBI patients whose PbtO(2) was monitored. The relationship between Hgb-categorized into four quartiles (≤9; 9-10; 10.1-11; >11 g/dl)-and PbtO(2) was analyzed using mixed-effects models. Anemia with compromised PbtO(2) was defined as episodes of Hgb ≤ 9 g/dl with simultaneous PbtO(2) < 20 mmHg. Outcome was assessed at 30 days using the Glasgow outcome score (GOS), dichotomized as favorable (GOS 4-5) vs. unfavorable (GOS 1-3). RESULTS: We analyzed 474 simultaneous Hgb and PbtO(2) samples from 80 patients (mean age 44 ± 20 years, median GCS 4 (3-7)). Using Hgb > 11 g/dl as the reference level, and controlling for important physiologic covariates (CPP, PaO(2), PaCO(2)), Hgb ≤ 9 g/dl was the only Hgb level that was associated with lower PbtO(2) (coefficient -6.53 (95 % CI -9.13; -3.94), p < 0.001). Anemia with simultaneous PbtO(2) < 20 mmHg, but not anemia alone, increased the risk of unfavorable outcome (odds ratio 6.24 (95 % CI 1.61; 24.22), p = 0.008), controlling for age, GCS, Marshall CT grade, and APACHE II score. CONCLUSIONS: In this cohort of severe TBI patients whose PbtO(2) was monitored, a Hgb level no greater than 9 g/dl was associated with compromised PbtO(2). Anemia with simultaneous compromised PbtO(2), but not anemia alone, was a risk factor for unfavorable outcome, irrespective of injury severity.

Dynamic changes in brain functional connectivity during concurrent dual-task performance.

This study investigated the spatial, spectral, temporal and functional proprieties of functional brain connections involved in the concurrent execution of unrelated visual perception and working memory tasks. Electroencephalography data was analysed using a novel data-driven approach assessing source coherence at the whole-brain level. Three connections in the beta-band (18-24 Hz) and one in the gamma-band (30-40 Hz) were modulated by dual-task performance. Beta-coherence increased within two dorsofrontal-occipital connections in dual-task conditions compared to the single-task condition, with the highest coherence seen during low working memory load trials. In contrast, beta-coherence in a prefrontal-occipital functional connection and gamma-coherence in an inferior frontal-occipitoparietal connection was not affected by the addition of the second task and only showed elevated coherence under high working memory load. Analysis of coherence as a function of time suggested that the dorsofrontal-occipital beta-connections were relevant to working memory maintenance, while the prefrontal-occipital beta-connection and the inferior frontal-occipitoparietal gamma-connection were involved in top-down control of concurrent visual processing. The fact that increased coherence in the gamma-connection, from low to high working memory load, was negatively correlated with faster reaction time on the perception task supports this interpretation. Together, these results demonstrate that dual-task demands trigger non-linear changes in functional interactions between frontal-executive and occipitoparietal-perceptual cortices.

Spatio-temporal Brain Dynamics Mediating Post-error Behavioral Adjustments.

Optimal behavior relies on flexible adaptation to environmental requirements, notably based on the detection of errors. The impact of error detection on subsequent behavior typically manifests as a slowing down of RTs following errors. Precisely how errors impact the processing of subsequent stimuli and in turn shape behavior remains unresolved. To address these questions, we used an auditory spatial go/no-go task where continual feedback informed participants of whether they were too slow. We contrasted auditory-evoked potentials to left-lateralized go and right no-go stimuli as a function of performance on the preceding go stimuli, generating a 2 × 2 design with "preceding performance" (fast hit [FH], slow hit [SH]) and stimulus type (go, no-go) as within-subject factors. SH trials yielded SH trials on the following trials more often than did FHs, supporting our assumption that SHs engaged effects similar to errors. Electrophysiologically, auditory-evoked potentials modulated topographically as a function of preceding performance 80-110 msec poststimulus onset and then as a function of stimulus type at 110-140 msec, indicative of changes in the underlying brain networks. Source estimations revealed a stronger activity of prefrontal regions to stimuli after successful than error trials, followed by a stronger response of parietal areas to the no-go than go stimuli. We interpret these results in terms of a shift from a fast automatic to a slow controlled form of inhibitory control induced by the detection of errors, manifesting during low-level integration of task-relevant features of subsequent stimuli, which in turn influences response speed.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin¿induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Cell locations AQP1, AQP4 and 9 in the non-human primate brain

Rapport de synthèse : La présence de trois canaux d'eau, appelés aquaporines AQP1, AQP4 et AQP9, a été observée dans le cerveau sain ainsi que dans plusieurs modèles des pathologies cérébrales des rongeurs. Peu est connu sur la distribution des AQP dans le cerveau des primates. Cette connaissance sera utile pour des futurs essaies médicamenteux qui visent à prévenir la formation des oedèmes cérébraux. Nous avons étudié l'expression et la distribution cellulaire des AQP1, 4 et 9 dans le cerveau primate non-humain. La distribution des AQP4 dans le cerveau primate non-humain a été observée dans des astrocytes périvasculaires, comparable à l'observation faite dans le cerveau du rongeur. Contrairement à ce qui a été décrit chez le rongeur, l'AQPI chez le primate est exprimée dans les processus et dans les prolongations périvasculaires d'un sous-type d'astrocytes, qui est avant tout localisé dans la matière blanche et dans la glia limitans et qui est peut-être impliqué dans l'homéostasie de l'eau. L'AQPI a aussi été observée dans les neurones qui innervent des vaisseaux sanguins de la pie-mère, suggérant un rôle possible dans la régularisation de la vascularisation cérébrale. Comme décrit chez le rongeur, le mRNA et les protéines de l'AQP9 ont été détectés dans des astrocytes et dans des neurones catécholaminergiques. Chez le primate, des localisations supplémentaires ont été observées dans des populations de neurones placées dans certaines zones corticales. Cet article décrit une étude détaillée sur la distribution des AQP1, 4 et 9 dans le cerveau primate non-humain. Les observations faites s'additionnent aux data déjà publié sur le cerveau du rongeur. Ces importantes différences entre les espèces doivent être considérées dans l'évaluation des médicaments qui agiront potentiellement sur des AQP des primates non-humains avant d'entrer dans la phase des essais cliniques sur des humains.