Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.

Signal changes in the spinal cord of the rat after injection of formalin into the hindpaw: Characterization using functional magnetic resonance imaging

Changes in metabolism and local circulation occur in the spinal cord during peripheral noxious stimulation. Evidence is presented that this stimulation also causes signal intensity alterations in functional magnetic resonance images of the spinal cord during formalin-induced pain. These results indicate the potential of functional magnetic resonance imaging in assessing noninvasively the extent and intensity of spinal cord excitation in this well characterized pain model. Therefore, the aim of this study was to establish functional magnetic resonance imaging as a noninvasive method to characterize temporal changes in the spinal cord after a single injection of 50 μl of formalin subcutaneously into the hindpaw of the anesthetized rat. This challenge produced a biphasic licking activity in the freely moving conscious animal. Images of the spinal cord were acquired within 2 min, enabling monitoring of the site and the temporal evolution of the signal changes during the development of formalin-induced hyperalgesia without the need of any surgical procedure. The time course of changes in the spinal cord functional image in the isoflurane-anesthetized animal was similar to that obtained from behavioral experiments. Also, comparable physiological data, control experiments, and the inhibition of a response through application of the local anesthetic agent lidocaine indicate that the signal changes observed after formalin injection were specifically related to excitability changes in the relevant segments of the lumbar spinal cord. This approach could be useful to characterize different models of pain and hyperalgesia and, more importantly, to evaluate effects of analgesic drugs.

Detection of cortical activation during averaged single trials of a cognitive task using functional magnetic resonance imaging

Functional neuroimaging studies in human subjects using positron emission tomography or functional magnetic resonance imaging (fMRI) are typically conducted by collecting data over extended time periods that contain many similar trials of a task. Here methods for acquiring fMRI data from single trials of a cognitive task are reported. In experiment one, whole brain fMRI was used to reliably detect single-trial responses in a prefrontal region within single subjects. In experiment two, higher temporal sampling of a more limited spatial field was used to measure temporal offsets between regions. Activation maps produced solely from the single-trial data were comparable to those produced from blocked runs. These findings suggest that single-trial paradigms will be able to exploit the high temporal resolution of fMRI. Such paradigms will provide experimental flexibility and time-resolved data for individual brain regions on a trial-by-trial basis.

Retinotopic mapping of lateral geniculate nucleus in humans using functional magnetic resonance imaging

Subcortical nuclei in the thalamus, which play an important role in many functions of the human brain, provide challenging targets for functional mapping with neuroimaging techniques because of their small sizes and deep locations. In this study, we explore the capability of high-resolution functional magnetic resonance imaging at 4 Tesla for mapping the retinotopic organization in the lateral geniculate nucleus (LGN). Our results show that the hemifield visual stimulation only activates LGN in the contralateral hemisphere, and the lower-field and upper-field visual stimulations activate the superior and inferior portion of LGN, respectively. These results reveal a similar retinotopic organization between the human and nonhuman primate LGN and between LGN and the primary visual cortex. We conclude that high-resolution functional magnetic resonance imaging is capable of functional mapping of suborganizations in small nuclei together with cortical activation. This will have an impact for studying the thalamocortical networks in the human brain.

Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

A Cross-Polarization, Magic-Angle-Spinning, 13C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls1

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by 31P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before 31P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

The hippocampal formation participates in novel picture encoding: evidence from functional magnetic resonance imaging.

Considerable evidence exists to support the hypothesis that the hippocampus and related medial temporal lobe structures are crucial for the encoding and storage of information in long-term memory. Few human imaging studies, however, have successfully shown signal intensity changes in these areas during encoding or retrieval. Using functional magnetic resonance imaging (fMRI), we studied normal human subjects while they performed a novel picture encoding task. High-speed echo-planar imaging techniques evaluated fMRI signal changes throughout the brain. During the encoding of novel pictures, statistically significant increases in fMRI signal were observed bilaterally in the posterior hippocampal formation and parahippocampal gyrus and in the lingual and fusiform gyri. To our knowledge, this experiment is the first fMRI study to show robust signal changes in the human hippocampal region. It also provides evidence that the encoding of novel, complex pictures depends upon an interaction between ventral cortical regions, specialized for object vision, and the hippocampal formation and parahippocampal gyrus, specialized for long-term memory.

Dynamic contrast-enhanced magnetic resonance imaging reveals stress-induced angiogenesis in MCF7 human breast tumors.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space. Correlation of these maps with T2-weighted spin echo images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that surrounded necrotic loci. The high leakage from the intratumoral permeable capillaries indicated an induction of a specific angiogenic process associated with stress conditions that cause necrosis. This induction was augmented in tumors responding to tamoxifen treatment. Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

Magnetic resonance imaging (MRI) detection of the murine brain response to light: temporal differentiation and negative functional MRI changes.

Using a 9.4 T MRI instrument, we have obtained images of the mouse brain response to photic stimulation during a period between deep anesthesia and the early stages of arousal. The large image enhancements we observe (often >30%) are consistent with literature results extrapolated to 9.4 T. However, there are also two unusual aspects to our findings. (i) The visual area of the brain responds only to changes in stimulus intensity, suggesting that we directly detect operations of the M visual system pathway. Such a channel has been observed in mice by invasive electrophysiology, and described in detail for primates. (ii) Along with the typical positive response in the area of the occipital portion of the brain containing the visual cortex, another area displays decreased signal intensity upon stimulation.

Activation of single whisker barrel in rat brain localized by functional magnetic resonance imaging.

The previously established cortical representation of rat whiskers in layer IV of the cortex contains distinct cylindrical columns of cellular aggregates, which are termed barrels and correlate in a one-to-one relation to whiskers on the contralateral rat face. In the present study, functional magnetic resonance imaging (fMRI) of the rat brain was used to map whisker barrel activation during mechanical up-down movement (+/- 2.5 mm amplitude at 8 Hz) of single/multiple whisker(s). Multislice gradient echo fMRI experiments were performed at 7 T with in-plane image resolution of 220 x 220 microns, slice thickness of 1 mm, and echo time of 16 ms. Highly significant (P < 0.001) and localized contralateral regions of activation were observed upon stimulation of single/multiple whisker(s). In all experiments (n = 10), the locations of activation relative to bregma and midline were highly correlated with the neuroanatomical position of the corresponding whisker barrels, and the results were reproducible intra- and interanimal. Our results indicate that fMRI based on blood oxygenation level-dependent image contrast has the sensitivity to depict activation of a single whisker barrel in the rat brain. This noninvasive technique will supplement existing methods in the study of rat barrel cortex and should be particularly useful for the long-term investigations of central nervous system in the same animal.