Biomedical subjects

R F Hevner

Publications and source records attributed to R F Hevner.

11 recordsLinked to original sources

PubMed1995

A metabolic map of cytochrome oxidase in the rat brain: histochemical, densitometric and biochemical studies.

To examine brain patterns of metabolic and functional activity, the distribution of cytochrome oxidase, a mitochondrial enzyme marker for neuronal functional activity, was mapped throughout the rat brain. Mapping was done qualitatively by enzyme histochemistry of brain sections cut in three planes (coronal, sagittal and horizontal), and quantitatively by optical densitometry of stained sections and by biochemical assays of brain tissue homogenates. Activity of the enzyme was distributed in characteristic patterns and amounts that differed among various neural pathways, brain nuclei, cerebral cortical areas and layers, and neuron types. Gray matter essentially always had higher enzyme activity than did white matter, by a factor of eight- to 12-fold. Among different neural pathways, cytochrome oxidase activity was relatively high in special sensory, somatosensory and motor systems, and was relatively low in associative, limbic, autonomic and visceral regulatory systems (though exceptional areas were present). Among 11 different neuron types, nearly a two-fold range of histochemical staining intensities was observed, with the darkest staining in neurons of the mesencephalic trigeminal nucleus. The observed patterns of cytochrome oxidase activity were mostly similar to the patterns of 2-deoxyglucose uptake seen previously [Schwartz W. J. and Sharp F. R. (1978) J. comp. Neurol. 177, 335-360; Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916] in conscious, "resting" animals, though some differences were found. For example, whereas 2-deoxyglucose uptake was about three-fold higher in gray matter than in white matter [Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916], cytochrome oxidase activity was about eight- to 12-fold higher. This and other discrepancies probably reflect basic technical differences between these two methods. Compared to 2-deoxyglucose, cytochrome oxidase is more specific for oxidative rather than glycolytic metabolism, and more reflective of overall neuronal functional activity occurring over longer time periods lasting hours to weeks, rather than minutes. The anatomical resolution of cytochrome oxidase histochemistry is also finer than that of 2-deoxyglucose autoradiography, extending to the electron microscopic level. The metabolic map of cytochrome oxidase activity reveals patterns of normal brain function, and may be useful as a baseline for comparison in studies of brain disease, development, ageing and plasticity.

Animals↗

PubMed1993

An optimized method for determining cytochrome oxidase activity in brain tissue homogenates.

We have developed a method to accurately and reproducibly determine the total activity of cytochrome oxidase (CO) in rat brain tissue homogenates. Previously, accurate measurements have been difficult to obtain because detergents, which are needed to disrupt membranes and unmask CO, also inhibit the enzyme by solubilizing certain phospholipids required for rapid turnover. We compared various methods of sample preparation, and found that maximal CO activity in homogenates could be obtained using specific concentrations of detergents. The range of optimal detergent concentrations was relatively narrow, as CO activity fell sharply with small deviations from the optimum. Of 5 detergents tested, deoxycholate stimulated CO maximally over the widest range of concentrations. In deoxycholate-treated homogenate samples, the calculated CO turnover number was about 480 s-1, indicating that overall enzyme activity was maximal or near maximal, and therefore that the total content of CO was probably detected. This method was reproducible with large or small samples (e.g., < 1 mg tissue), and should be applicable to studies of neural tissue in general.

Animals↗

PubMed1993

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Animals↗

PubMed1993

Mitochondrial and nuclear gene expression for cytochrome oxidase subunits are disproportionately regulated by functional activity in neurons.

Mitochondrial respiratory complexes such as cytochrome oxidase (CO) contain both mitochondrial- and nuclear-encoded subunits. To determine whether mitochondrial and nuclear gene expression are regulated proportionately in neurons, we analyzed CO subunit mRNA and mitochondrial DNA (mtDNA) levels by in situ hybridization and grain counting in the visual system of normal and monocular TTX-treated monkeys. We compared the regulation of these molecules with the regulation of CO activity and CO protein, analyzed by histochemistry and immunohistochemistry, respectively. In normal animals, CO activity was in general related more closely to mtDNA and CO subunit I (COI) (mitochondrial-encoded) mRNA levels than to COIV or COVIII (nuclear-encoded) mRNA levels. For example, puffs (also known as blobs) of high CO activity in striate cortex were enriched in mtDNA and COI mRNA, but not COIV or COVIII mRNA. In 3-7 d TTX-treated animals, proportionate decreases in CO activity and CO protein were observed in specific visual centers; these changes were accompanied by disproportionate decreases in COI, COIV, and COVIII mRNA levels. After 7 d of TTX, COI mRNA fell by 49 +/- 3% (mean +/- SEM) in LGN neurons, while COIV and COVIII mRNAs fell by only 18 +/- 3% and 29 +/- 3%, respectively. In comparison, CO activity decreased by 23 +/- 2%, and mtDNA by 26 +/- 4%. Qualitative observations in striate cortex also indicated that COI mRNA changed more than COIV mRNA, COVIII mRNA, mtDNA, or CO activity. Our results suggest that the local distribution of CO within neurons, and acute regulatory changes in CO activity occurring over periods of days are controlled mainly by regulation of the mitochondrial genes that encode the catalytic subunits of the enzyme.

Animals↗

PubMed1992

Entorhinal cortex of the human, monkey, and rat: metabolic map as revealed by cytochrome oxidase.

The entorhinal cortex (EC) is a medial temporal lobe area involved in memory consolidation. Results from previous studies suggest that the upper layers of the EC may be organized into anatomical-neurochemical modules associated with pathways through the neuron clusters in layers II and III. To study metabolic patterns in the EC and to look for correlates of the proposed modules, we examined the distribution of cytochrome oxidase (CO) in the human, monkey, and rat EC. CO is a mitochondrial enzyme that has been used to study modules in other cortical areas. In all three species, the neuron clusters in layers II-III were darkly CO-reactive, whereas most of the neuropil between clusters was lightly or moderately CO-reactive. However, some neuropil regions directly adjacent to the neuron clusters were also darkly CO-reactive, especially in the human; these neuropil areas included portions of layers I and II. In tangential sections through layers I-II, the areas of dark staining formed a consistent pattern, comprised of partially interconnected islands and stripes associated with the neuron clusters. In the EC from one human hemisphere, approximately 200-250 CO-reactive layer II islands were present. EC layers other than I-III also showed characteristic CO staining intensities, but no evidence of modularity. Our results indicate that CO staining labels distinct compartments related to the neuron clusters in the upper EC layers. We propose that these compartments may represent modules for cortical processing, analogous to the CO-labeled modules in some other areas of cortex.

Animals↗

PubMed1992

Coordination of ATP production and consumption in brain: parallel regulation of cytochrome oxidase and Na+, K(+)-ATPase.

Previous studies have shown that most neuronal ATP is produced by oxidative metabolism, and consumed by Na+,K(+)-ATPase. We hypothesized that the distribution of Na+,K(+)-ATPase in brain would correlate with that of the energy-producing enzyme, cytochrome oxidase (CO). We localized these enzymes in monkey hippocampus and striate cortex by histochemistry and immunohistochemistry. Their distributions were generally similar, although some differences were observed. We also studied regulation of enzyme levels, using monocular impulse blockage with tetrodotoxin (TTX) to alter neuronal activity in the visual system. Parallel changes in CO and Na+,K(+)-ATPase activity were induced in striate cortex. These results provide further evidence that neuronal energy demands regulate CO levels and distribution.

Adenosine Triphosphate↗

PubMed1991

Neuronal expression of nuclear and mitochondrial genes for cytochrome oxidase (CO) subunits analyzed by in situ hybridization: comparison with CO activity and protein.

Cytochrome oxidase (CO) is a mitochondrial energy-generating enzyme of the oxidative phosphorylation pathway. In neurons, CO activity varies among different cells and compartments (perikarya, dendrites, axons, and terminals) according to their physiological activity and metabolic requirements. Regulation of enzyme protein levels, rather than enzyme turnover number, largely accounts for local variations in CO activity (Hevner and Wong-Riley, 1989, 1990). In the present study, we examined how CO activity and protein levels are related to mitochondrial DNA (mtDNA) and CO subunit mRNA levels in neurons and neuronal compartments. Mammalian CO comprises 13 subunits (Kadenbach et al., 1983), of which three are encoded in mtDNA and 10 in nuclear genes. We studied one mitochondrial-encoded mRNA [subunit I (COI)], two nuclear-encoded mRNAs (COIV, COVIII), and mtDNA, using in situ hybridization to determine their distributions in monkey hippocampus, cerebellum, and primary visual cortex. We compared their distributions with those of CO activity and protein, determined by histochemistry and immunohistochemistry, respectively. In all regions, the local content of mtDNA was similar, but not identical, to the activity and amount of CO. Expression of COI mRNA was not proportional to mtDNA abundance or CO activity and protein, but instead was highest in cell bodies, lower in dendrites, and undetectable in axon terminals. COIV and COVIII mRNAs were detected exclusively in perikarya and proximal dendrites. Thus, the nuclear-encoded subunits of CO are probably translated mainly in neuronal cell bodies and allocated to other compartments posttranslationally. Regulation of CO was studied in two monkeys treated by monocular tetrodotoxin (TTX) injection, a procedure that blocks impulses from one eye. In those animals, cortical changes in CO activity were correlated with changes in mtDNA and in COI, COIV, and COVIII mRNA. Our results suggest that neuronal CO is synthesized and assembled mainly in cell bodies and indicate that both nuclear and mitochondrial CO subunit genes are regulated by neuronal activity.

Animals↗

PubMed1990

Regulation of cytochrome oxidase protein levels by functional activity in the macaque monkey visual system.

The activity of cytochrome oxidase (CO), a mitochondrial enzyme of oxidative energy metabolism, is regulated by nerve cell functional activity. The mechanism of CO activity regulation was studied using histochemical and immunohistochemical methods to show the distributions of CO activity and protein, respectively, in the macaque monkey visual system under normal and experimental conditions. In normal animals, patterns of CO activity were found to reflect underlying patterns of CO protein distribution; for example, puffs of high CO activity in cortical area 17 contained high levels of CO protein. Experimental animals were injected monocularly with TTX for 3-4 weeks; this treatment blocks retinal impulses in the injected eye and results in decreased CO activity in lateral geniculate laminae and striate cortical columns normally driven by the treated eye. The experimentally induced decreases in CO activity were also found to reflect underlying parallel decreases in CO protein levels. These results suggest that CO activity is regulated mainly at the level of the local amount rather than the turnover number of the enzyme and imply that the rates of CO synthesis and/or degradation are regulated by neural functional activity.

Animals↗

PubMed1989

Double labeling of cytochrome oxidase and gamma-aminobutyric acid in central nervous system neurons of adult cats.

The relationship between the levels of cytochrome oxidase and gamma-aminobutyric acid (GABA) was investigated within single neurons by double labeling the 2 markers in the same section. Double staining was equally effective when immunogold-silver staining of GABA was followed by indirect immunoperoxidase labeling of cytochrome oxidase, or when cytochrome oxidase histochemistry was followed by immunogold-silver staining of GABA. Neurons in the perigeniculate nucleus (PGN) and basket cell terminals in the cerebellum were GABA positive and rich in cytochrome oxidase. Interneurons of the lateral geniculate nucleus (LGN) as well as stellate and Golgi cells of the cerebellum were GABA-rich but poor in cytochrome oxidase. These results demonstrate that there is no consistent relationship between the levels of cytochrome oxidase and GABA in neurons.

Animals↗

PubMed1989

Brain cytochrome oxidase: purification, antibody production, and immunohistochemical/histochemical correlations in the CNS.

Cytochrome oxidase (CO) is a mitochondrial energy-generating enzyme used in brain studies as a marker of neural functional activity. The activity of CO in different brain regions, revealed histochemically, is distributed nonhomogeneously but in distinct patterns. Localized differences in CO activity could arise from localized differences in enzyme amount or from localized regulation of enzyme turnover number (molecular activity). To distinguish between these alternatives, we used antibodies against purified calf brain CO to assess the immunohistochemical distribution of CO amount (protein immunoreactivity) in several brain regions. Calf brain mitochondria (synaptic and nonsynaptic populations) were isolated from gray matter homogenates by differential centrifugation. CO was purified from detergent extracts of the mitochondria by cytochrome c-Sepharose 4B affinity chromatography. Antisera against the purified CO were raised in rabbits. The antibodies reacted specifically with CO, predominantly subunit IV, in SDS immunoblots. The antibodies did not react in SDS immunoblots with any other proteins solubilized from mitochondria or caudate nucleus but did cross-react with brain CO from other mammalian species and with bovine heart CO. The immunohistochemical distribution of CO amount matched the histochemical distribution of CO activity in all regions tested, including the monkey hippocampus and the mouse olfactory bulb, somatosensory (barrel) cortex, and cerebellum. Thus, the amount of CO in neural tissue is distributed in the same nonhomogeneous pattern as the histochemical activity of CO. The results suggest that mechanisms exist by which CO molecules are selectively distributed within neurons to meet local metabolic demands posed by neural functional activity.

Animals↗

PubMedDate not supplied

Cytochrome oxidase in the human visual cortex: distribution in the developing and the adult brain.

Cytochrome-oxidase (CO) histochemistry has revealed important functional subdivisions, modules, and processing streams in the macaque visual cortex. The present study is aimed at analyzing the development and characteristics of CO patterns in the human visual cortex by means of histochemistry and immunohistochemistry. At 26 weeks of gestation, both the ventricular and subventricular zones had low levels of CO, while the cortical plate had moderate levels of CO. At birth, supragranular CO-rich zones (puffs) were not clearly organized, indicating that the development of puffs in the neonatal striate cortex lags behind that of the macaque monkey, whose puffs appear weeks before birth. Puffs were more clearly discernible in human cortex at postnatal day 24, and became well organized by the fourth postnatal month. Layer IVc alpha in the neonate exhibited a higher level of activity and amount of CO than the central portion of IVc beta, which contained a dense aggregate of small neurons. The base of IVc beta, however, was often as CO reactive as IVc alpha. In contrast, the majority of specimens available to us from the fourth postnatal month and from adults with no known neurological diseases had significantly greater CO reactivity in layer IVc beta than in IVc alpha. Layer VI was moderately reactive for CO throughout development. In V2, stripes with globular zones of high CO activity were sporadically present at birth, suggesting that their development may parallel or precede that of puffs in V1. These stripes with CO-rich globular zones became more prominent in the adult and radiated orthogonally from the V1/V2 border. They were not, however, clearly organized into alternating thick and thin stripes as they are in the squirrel monkey. Visual cortical areas beyond V2 exhibited high CO activity mainly in layers III and IV and moderate levels in VI, suggesting that sites associated with cortico-cortical pathways may be metabolically most active.

Adult↗