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PubMed1995

Morphological alterations in neocortical and cerebellar GABAergic neurons in a canine model of juvenile Batten disease.

The pathogenesis of brain dysfunction in a canine model of juvenile Batten disease was studied with techniques designed to determine sequential changes in mitochondrial morphology and cytochrome oxidase (CO) activity, and in neurons and synapses using gamma-aminobutyric acid (GABA) as a neurotransmitter. Histochemical and immunocytochemical methods were employed. Mitochondrial alterations were found in a select population of nonpyramidal neurons in neocortex and claustrum, and in cerebellar basket cells. Proportions of affected neurons at any one time remained constant over the disease course, with morphologically-abnormal mitochondria first being recognized at age 6 months. Enlarged mitochondria were readily identifiable at the light microscope (LM) level as large CO-positive or mitochondrial antibody-positive granular structures. Colabelling with antibodies to GABA or to parvalbumin (PV) indicated that most of these cells were GABAergic. Ultrastructurally, atypical mitochondria were characterized by globular enlargement, intramitochondrial membranous inclusions, and disorganized internal structure. CO activity in all other cell somata and in neuropil was diminished compared with normal, age-matched tissue. Glutamic acid decarboxylase (GAD), PV, and GABA studies demonstrated loss of GABAergic neurons and synapses in cortex and cerebellum of affected dogs. These results indicate that abnormal mitochondria are present in neurons in Batten disease, and suggest that suboptimal mitochondrial function may play a role in the pathogenic mechanisms of brain dysfunction in this disorder.

Animals↗
PubMed1985

An analysis of the cellular localization of cytochrome oxidase in the lateral geniculate nucleus of the adult cat.

The distribution of cytochrome oxidase (C.O.) was examined in the normal adult cat lateral geniculate nucleus at the cellular and electron-microscopic levels. The darker reactivity of the X- and/or Y-receptive laminae (A, A1, magnocellular lamina C [Cm], and medial interlaminar nucleus [MIN]) compared with the lightly reactive W-receptive parvicellular lamina C (Cp) indicates that there are pathway-specific histochemical differences in the visual system of the cat. At the cellular level, darkly reactive large cells in the lateral geniculate nucleus (LGN) closely resemble class 1, Y-cells, in relative size and distribution, thus indicating that C.O. histochemistry may be used as a functional marker for these cells. Perigeniculate neurons are also darkly reactive. Neuronal classes 2, 4, and 3 (presumed X-cells, W-cells, and/or interneurons) have moderate to lightly reactive perikarya. The darkly reactive neuronal classes tend to receive relatively stronger proximal excitatory synaptic input than do the less reactive neuronal classes. Since all neuronal classes appeared to have darkly (or moderately) reactive dendrites, C.O. reactivity must differ between dendrite and soma of some neuronal classes. At the electron-microscopic level, distinct components of the neuropil tend to have specific levels of C.O. reactivity. The predominance of darkly reactive mitochondria in dendrites indicates that dendrites are metabolically very active. RLD and may F's, but few large axon terminals with round vesicles (RL) or small axon terminals with round vesicles (RS) profiles are darkly reactive, implying that specific classes of presynaptic structures are more active than others. Thus C.O. histochemistry may be useful for distinguishing not only functionally active neuronal classes such as Y-cells and perigeniculate (PG) neurons from less active neuronal classes, but also functionally more or less active parts of the same neuron including its dendrites, axons, and/or axon terminals.

Animals↗
PubMed1986

Differential effect of visual deprivation on cytochrome oxidase levels in major cell classes of the cat LGN.

Cytochrome oxidase histochemistry was used to examine the effects of visual deprivation on the development of neurons in the lateral geniculate nucleus of the kitten. Early postnatal monocular suture results in a decrease in reactivity within the neuropil of visually deprived binocular laminae A, A1, magnocellular C, and medial interlaminar nucleus. Within these regions, monocular suture has a greater effect on the relative numbers of, and the growth of darkly reactive (normally large), presumed Y-cells than on other less reactive geniculate neuronal classes. The decreases in the reactivity of the neuropil may be attributed to the decreases in the number of mitochondria, the number of darkly reactive mitochondria, and/or the number of darkly reactive mitochondria localized within dendrites. Although all classes of dendrites appear to be adversely affected, the decrease in C.O. reactivity was most dramatic within the presumed proximal dendrites of class 1 Y-cells. These dendrites were identified by the type of synaptic contacts they formed with retinal terminals (Rapisardi and Miles, '84, J. Comp. Neurol. 223:515-534; Wilson et al., '84, Proc. R. Soc. Lond. [Biol.] 221:411-436). As with Y-cells, the effects of monocular suture on the large darkly reactive cells were not as dramatic at sites where binocular interactions were either absent or where they had been experimentally eliminated. Based on the present and previously reported findings from several laboratories, it is likely that the selective physiological and morphological effects of monocular suture on Y-cells are accompanied by metabolic deficits involving both dendrites and perikarya. These effects appear to be due more to binocular interactions than to visual deprivation per se.

Animals↗
PubMed1995

Double labeling of GABA and cytochrome oxidase in the macaque visual cortex: quantitative EM analysis.

In the primate striate cortex, cytochrome oxidase (CO)-rich puffs differ from CO-poor interpuffs in their metabolic levels and physiological properties. The neurochemical basis for their metabolic and physiological differences is not well understood. The goal of the present study was to examine the relationship between the distribution of gamma aminobutyric acid (GABA)/non-GABA synapses and CO levels in postsynaptic neuronal profiles and to determine whether or not a difference existed between puffs and interpuffs. By combining CO histochemistry and postembedding GABA immunocytochemistry on the same ultrathin sections, the simultaneous distribution of the two markers in individual neuronal profiles was quantitatively analyzed. In both puffs and interpuffs, GABA-immunoreactive (GABA-IR) neurons were the only cell type that received both non-GABA-IR (presumed excitatory) and GABA-IR (presumed inhibitory) axosomatic synapses, and they had three times as many mitochondria darkly reactive for CO than non-GABA-IR neurons, which received only GABA-IR axosomatic synapses. GABA-IR neurons and terminals in puffs had a larger mean size, about twice as many darkly reactive mitochondria, and a higher ratio of non-GABA-IR to GABA-IR axosomatic synapses than those in interpuffs (2.3:1 vs. 1.6:1; P < 0.01). There were significantly more synapses of both non-GABA-IR and GABA-IR types in the neuropil of puffs than of interpuffs; however, the ratio of non-GABA-IR to GABA-IR synapses was significantly higher in puffs (2.86:1) than in interpuffs (2.08:1; P < 0.01). Our results are consistent with the hypothesis that the level of oxidative metabolism in postsynaptic neurons and neuronal processes is tightly governed by the strength and proportion of excitatory over inhibitory synapses. Thus, the present results suggest that (1) GABA-IR neurons in the macaque striate cortex have a higher level of oxidative metabolism than non-GABA ones because their somata receive direct excitatory synapses and their terminals are more tonically active; (2) the higher proportion of presumed excitatory synapses in puffs imposes a greater energy demand there than in interpuffs; and (3) excitatory synaptic activity may be more prominent in puffs than in interpuffs because puffs receive a greater proportion of excitatory synapses from multiple sources including the lateral geniculate nucleus, which is not known to project to the interpuffs.

Animals↗
PubMed1993

Effects of development and altered gravity conditions on cytochrome oxidase activity in a vestibular nucleus of the larval teleost brain: a quantitative electronmicroscopical study.

The mitochondrial enzyme, cytochrome oxidase, was localized cytochemically in the nucleus magnocellularis, a primary relay nucleus of vestibular information within the area octavolateralis in the fish brain. Larvae of the cichlid fish Oreochromis mossambicus were analyzed at different developmental stages (4, 10, and 35 days post-hatching) and after long-term exposure (8 days) to increased gravity (2-4 g). Quantification of highly reactive, moderately reactive, and nonreactive mitochondria reveals differences in the cytochrome oxidase activity of various cellular structures, for example, perikarya of neurons, presynaptic terminals, and myelinated and nonmyelinated cell profiles. Cytochrome oxidase activity in the mitochondria of neuronal perikarya increases during development which parallels the differentiation of the area octavolateralis. This possibly reflects the increasing energy demand during maturation and innervation of the magnocellular nucleus. Hyper-g-exposure of the larvae for 8 days (centrifuge) caused a further augmentation of cytochrome oxidase activity in the perikarya within the nucleus magnocellularis. This may reflect an increased oxidative metabolism resulting from the need for compensation of altered inputs from gravity-sensitive epithelia in the inner ear. Another possibility is that acceleration within a centrifuge causes physiological stress for the animals and, therefore, influences the cytochrome oxidase activity in neurons.

Animals↗
PubMed1994

Subfractional analysis of cyanobacterial membranes and isolation of plasma membranes by aqueous polymer two-phase partitioning.

The present work demonstrates that partition in aqueous polymer two-phase systems offers a rapid method for separation and isolation of thylakoid, plasma, and outer membranes from cyanobacteria. Pure plasma membranes from Phormidium laminosum can be isolated by this method within 3 h, starting with total membranes obtained by French press treatment of the cyanobacterial cells. The isolated plasma membranes have a broad density profile, giving rise to three subpopulations. The main fraction has the same density as the abundant thylakoid membranes. This fraction has not been resolved in previous separations based on sucrose gradient centrifugation, which is the only method previously used for isolation of cyanobacterial plasma membranes. Another advantage of the aqueous polymer two-phase system is that it can handle large quantities of starting material, which is essential to obtain a satisfactory yield since plasma membranes constitute only a very small fraction of the total membrane content in a cyanobacterial cell. The isolation procedure results in a pure plasma membrane preparation with retained cytochrome c oxidase activity. The results also point to the possibility of a lateral heterogeneity in the organization of the cyanobacterial plasma membrane.

Cell Membrane↗
PubMed1994

Cytochrome c oxidase in proteoliposomes visualised by platinum-carbon and by tungsten-tantalum shadowing: image analysis.

Beef heart cytochrome c oxidase complexes incorporated into phospholipid liposomes were examined by freeze-fracture electron microscopy. Enzyme molecules are inserted into the membrane asymmetrically, with larger projections on the 'C' side, where cytochrome c binding occurs, than on the 'M' (matrix-facing) side. Visualisation of the complexes was improved by: (i) image analysis, to determine details of size and shape, and (ii) tungsten-tantalum (W/Ta) rather than platinum-carbon (Pt/C) shadowing, which permits examination of smaller entities. Enzyme molecules are incorporated as dimers in the proteoliposomes. Some surface structural details of the embedded molecules can be discerned. Around each complex is seen a small area of modified lipid, the frozen annulus whose existence has been predicted with other methods.

Animals↗
PubMed1988

Cytochrome oxidase histochemistry in the rat hippocampus. A quantitative methodological study.

The diaminobenzidine (DAB) method was adapted for the microphotometric determination of cytochrome c oxidase (cyt ox) in the rat hippocampus. The qualitative and quantitative investigations at the light microscopic level showed that acetone and cytochrome c pretreatment of cryostat sections resulted in a significant increase of demonstrable cyt ox activities. The final incubation medium consisted of 7.5 mM DAB, 2% polyvinylalcohol (PVA) and 6% dimethyl sulfoxide in 0.1 M Hepes buffer; final pH 7.5. PVA was used to keep DAB and artificially oxidized DAB in solution. In the kinetic and endpoint measurements a linear response of the reaction with highest slope was observed only in the initial 5-6 min of reaction. Thereafter the slope decreased. Ultracytochemical demonstrations, which were performed as a topochemical control, showed reaction product only in mitochondria (cristae and intermembranous space). In contrast to vibratome sections all mitochondria reacted positively in cryostat sections of aldehyde-fixed hippocampi. The enhancement of reaction after acetone pretreatment of cryostat sections (light microscopic level) and after a freezing step in ultracytochemistry is discussed in connection with diffusion problems of DAB through mitochondrial membranes.

3,3'-Diaminobenzidine↗
PubMed1992

AZT-induced mitochondrial myopathy.

Histochemical, electron microscopy and biochemical studies were performed on muscle biopsy specimens from 11 AIDS patients treated with zidovudine. A peculiar association of structural abnormalities and mitochondrial dysfunction was found. Focal cytochrome c oxidase (COX) deficiency was evident in muscle sections from 9 patients, 8 of whom had received long-term treatment while one had been treated for 1 month only. Electron microscopy showed changes in number, size and structure of mitochondria. Biochemical studies proved partial COX and succinate cytochrome c reductase (SCR) deficiency in 4 patients; one patient had only reduced SCR activity. Our data confirm that AZT therapy can cause toxic myopathy with mitochondrial dysfunction.

Adult↗