Olfactory coding in the antennal lobe of the bumble bee Bombus terrestris.

Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honey bees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe of the bumble bee Bombus terrestris. Our results show that odorants induce reproducible neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecule's chemical structure, i.e. its carbon chain length and functional group. In addition, pairwise similarity among odor representations are conserved in bumble bees and honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

Assessment of the structural complexity of diffusion MRI voxels using 3D electron microscopy in the rat brain.

Validation and interpretation of diffusion magnetic resonance imaging (dMRI) requires detailed understanding of the actual microstructure restricting the diffusion of water molecules. In this study, we used serial block-face scanning electron microscopy (SBEM), a three-dimensional electron microscopy (3D-EM) technique, to image seven white and grey matter volumes in the rat brain. SBEM shows excellent contrast of cellular membranes, which are the major components restricting the diffusion of water in tissue. Additionally, we performed 3D structure tensor (3D-ST) analysis on the SBEM volumes and parameterised the resulting orientation distributions using Watson and angular central Gaussian (ACG) probability distributions as well as spherical harmonic (SH) decomposition. We analysed how these parameterisations described the underlying orientation distributions and compared their orientation and dispersion with corresponding parameters from two dMRI methods, neurite orientation dispersion and density imaging (NODDI) and constrained spherical deconvolution (CSD). Watson and ACG parameterisations and SH decomposition captured well the 3D-ST orientation distributions, but ACG and SH better represented the distributions due to its ability to model asymmetric dispersion. The dMRI parameters corresponded well with the 3D-ST parameters in the white matter volumes, but the correspondence was less evident in the more complex grey matter. SBEM imaging and 3D-ST analysis also revealed that the orientation distributions were often not axially symmetric, a property neatly captured by the ACG distribution. Overall, the ability of SBEM to image diffusion barriers in intricate detail, combined with 3D-ST analysis and parameterisation, provides a step forward toward interpreting and validating the dMRI signals in complex brain tissue microstructure.

Brain structure mediates the association between socioeconomic status and attention-deficit/hyperactivity disorder.

Low socioeconomic status (SES) is associated with greater risk for symptoms of attention-deficit/hyperactivity disorder (ADHD). One mechanism through which SES may confer risk for ADHD is by influencing brain structure. Alterations to cortical thickness, surface area and subcortical volume have been associated with low SES and with the presence of ADHD across multiple studies. The current study examined whether cortical thickness, surface area or subcortical volume mediate the associations between SES and ADHD in youth 3-21 years old (N = 874) from the Pediatric Imaging, Neurocognition and Genetics Study. Freesurfer was used to estimate cortical thickness, surface area and subcortical volume from structural magnetic resonance imaging. Parents reported on demographics, family SES, ADHD diagnoses and the presence of child attention problems. Statistical mediation was assessed using a bootstrap resampling procedure. Controlling for parental ADHD, child age, gender, birth weight and scanner, children in low SES families were more likely to be in the ADHD group. Consistent with previous reports in this sample, low SES was associated with reduced surface area across the frontal lobe and reduced subcortical volume in the amygdala, cerebellum, hippocampus and basal ganglia. Of these regions, a significant indirect effect of SES on ADHD status through subcortical volume was observed for the left cerebellum (95% confidence interval: 0.004, 0.022), the right cerebellum (95% confidence interval: 0.006, 0.025), and the right caudate (95% confidence interval: 0.002, 0.022). Environmentally mediated changes in the cerebellum and the caudate may be neurodevelopmental mechanisms explaining elevated risk of ADHD in children in low SES families.

Acute Toxicity Assessment: Macroscopic and Ultrastructural Effects in Mice Treated with Oral Tetrodotoxin.

Tetrodotoxin (TTX) is an extremely toxic marine compound produced by different genera of bacteria that can reach humans through ingestion mainly of pufferfish but also of other contaminated fish species, marine gastropods or bivalves. TTX blocks voltage-gated sodium channels inhibiting neurotransmission, which in severe cases triggers cardiorespiratory failure. Although TTX has been responsible for many human intoxications limited toxicological data are available. The recent expansion of TTX from Asian to European waters and diversification of TTX-bearing organisms entail an emerging risk of food poisoning. This study is focused on the acute toxicity assessment of TTX administered to mice by oral gavage following macroscopic and microscopic studies. Necropsy revealed that TTX induced stomach swelling 2 h after administration, even though no ultrastructural alterations were further detected. However, transmission electron microscopy images showed an increase of lipid droplets in hepatocytes, swollen mitochondria in spleens, and alterations of rough endoplasmic reticulum in intestines as hallmarks of the cellular damage. These findings suggested that gastrointestinal effects should be considered when evaluating human TTX poisoning.

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

Assessment of a nutritional supplement containing resveratrol, prebiotic fiber, and omega-3 fatty acids for the prevention and treatment of mild traumatic brain injury in rats.

Children and adolescents have the highest rates of traumatic brain injury (TBI), with mild TBI (mTBI) accounting for most of these injuries. Adolescents are particularly vulnerable and often suffer from post-injury symptomologies that may persist for months. We hypothesized that the combination of resveratrol (RES), prebiotic fiber (PBF), and omega-3 fatty acids (docosahexaenoic acid (DHA)) would be an effective therapeutic supplement for the mitigation of mTBI outcomes in the developing brain. Adolescent male and female Sprague-Dawley rats were randomly assigned to the supplement (3S) or control condition, which was followed by a mTBI or sham insult. A behavioral test battery designed to examine symptomologies commonly associated with mTBI was administered. Following the test battery, tissue was collected from the prefrontal cortex (PFC) and primary auditory cortex for Golgi-Cox analysis of spine density, and for changes in expression of 6 genes (Aqp4, Gfap, Igf1, Nfl, Sirt1, and Tau). 3S treatment altered the behavioral performance of sham animals indicating that dietary manipulations modify premorbid characteristics. 3S treatment prevented injury-related deficits in the longer-term behavior measures, medial prefrontal cortex (mPFC) spine density, and levels of Aqp4, Gfap, Igf1, Nfl, and Sirt1 expression in the PFC. Although not fully protective, treatment with the supplement significantly improved post-mTBI function and warrants further investigation.

Including diffusion time dependence in the extra-axonal space improves in vivo estimates of axonal diameter and density in human white matter.

Axonal density and diameter are two fundamental properties of brain white matter. Recently, advanced diffusion MRI techniques have made these two parameters accessible in vivo. However, the techniques available to estimate such parameters are still under development. For example, current methods to map axonal diameters capture relative trends over different structures, but consistently over-estimate absolute diameters. Axonal density estimates are more accessible experimentally, but different modeling approaches exist and the impact of the experimental parameters has not been thoroughly quantified, potentially leading to incompatibility of results obtained in different studies using different techniques. Here, we characterise the impact of diffusion time on axonal density and diameter estimates using Monte Carlo simulations and STEAM diffusion MRI at 7 T on 9 healthy volunteers. We show that axonal density and diameter estimates strongly depend on diffusion time, with diameters almost invariably overestimated and density both over and underestimated for some commonly used models. Crucially, we also demonstrate that these biases are reduced when the model accounts for diffusion time dependency in the extra-axonal space. For axonal density estimates, both upward and downward bias in different situations are removed by modeling extra-axonal time-dependence, showing increased accuracy in these estimates. For axonal diameter estimates, we report increased accuracy in ground truth simulations and axonal diameter estimates decreased away from high values given by earlier models and towards known values in the human corpus callosum when modeling extra-axonal time-dependence. Axonal diameter feasibility under both advanced and clinical settings is discussed in the light of the proposed advances.

Accelerating fibre orientation estimation from diffusion weighted magnetic resonance imaging using GPUs.

With the performance of central processing units (CPUs) having effectively reached a limit, parallel processing offers an alternative for applications with high computational demands. Modern graphics processing units (GPUs) are massively parallel processors that can execute simultaneously thousands of light-weight processes. In this study, we propose and implement a parallel GPU-based design of a popular method that is used for the analysis of brain magnetic resonance imaging (MRI). More specifically, we are concerned with a model-based approach for extracting tissue structural information from diffusion-weighted (DW) MRI data. DW-MRI offers, through tractography approaches, the only way to study brain structural connectivity, non-invasively and in-vivo. We parallelise the Bayesian inference framework for the ball & stick model, as it is implemented in the tractography toolbox of the popular FSL software package (University of Oxford). For our implementation, we utilise the Compute Unified Device Architecture (CUDA) programming model. We show that the parameter estimation, performed through Markov Chain Monte Carlo (MCMC), is accelerated by at least two orders of magnitude, when comparing a single GPU with the respective sequential single-core CPU version. We also illustrate similar speed-up factors (up to 120x) when comparing a multi-GPU with a multi-CPU implementation.

Automated transmission-mode scanning electron microscopy (tSEM) for large volume analysis at nanoscale resolution.

Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm(2) (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm(2) (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of ∼100 µm(2) and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (∼45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.

A time and cost efficient approach to functional and structural assessment of living neuronal tissue.

In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.

Development of a brain MRI-based hidden Markov model for dementia recognition.

BACKGROUND: Dementia is an age-related cognitive decline which is indicated by an early degeneration of cortical and sub-cortical structures. Characterizing those morphological changes can help to understand the disease development and contribute to disease early prediction and prevention. But modeling that can best capture brain structural variability and can be valid in both disease classification and interpretation is extremely challenging. The current study aimed to establish a computational approach for modeling the magnetic resonance imaging (MRI)-based structural complexity of the brain using the framework of hidden Markov models (HMMs) for dementia recognition. METHODS: Regularity dimension and semi-variogram were used to extract structural features of the brains, and vector quantization method was applied to convert extracted feature vectors to prototype vectors. The output VQ indices were then utilized to estimate parameters for HMMs. To validate its accuracy and robustness, experiments were carried out on individuals who were characterized as non-demented and mild Alzheimer's diseased. Four HMMs were constructed based on the cohort of non-demented young, middle-aged, elder and demented elder subjects separately. Classification was carried out using a data set including both non-demented and demented individuals with a wide age range. RESULTS: The proposed HMMs have succeeded in recognition of individual who has mild Alzheimer's disease and achieved a better classification accuracy compared to other related works using different classifiers. Results have shown the ability of the proposed modeling for recognition of early dementia. CONCLUSION: The findings from this research will allow individual classification to support the early diagnosis and prediction of dementia. By using the brain MRI-based HMMs developed in our proposed research, it will be more efficient, robust and can be easily used by clinicians as a computer-aid tool for validating imaging bio-markers for early prediction of dementia.

Neuro-immune interactions in the dove brain.

Mast cells (MC) are of hematopoetic origin. Connective tissue type MCs are able to function in IgE dependent and independent fashion, change their phenotype according to the tissue environment. They are able to enter the brain under normal physiological conditions, and move into this compact tissue made of neurons. In doves MCs are found only in the medial habenula (MH) and their number is changing according to the amount of sex steroids in the body. MCs are able to synthesize and store a great variety of biologically active compounds, like transmitters, neuromodulators and hormones. They are able to secrete GnRH. With the aid of electron microscopy we were able to describe MC-neuron interactions between GnRH-positive MCs and neurons. Piecemeal degranulation (secretory vesicles budding off swollen and active granules) seems to be a very efficient type of communication between MCs and surrounding neurons. Different types of granular and vesicular transports are seen between GnRH-immunoreactive MCs and neurons in the MH of doves. Sometimes whole granules are visible in the neuronal cytoplasm, in other cases exocytotic vesicles empty materials of MC origin. Thus MCs might modulate neuronal functions. Double staining experiments with IP3-receptor (IP3R), Ryanodine-receptor (RyR) and serotonin antibodies showed active MC population in the habenula. Light IP3R-labeling was present in 64-97% of the cells, few granules were labeled in 7-10% of MCs, while strong immunoreactivity was visible in 1-2% of TB stained cells. No immunoreactivity was visible in 28-73% of MCs. According to cell counts, light RyR-positivity appeared in 27-52%, few granules were immunoreactive in 4-19%, while strong immunopositivity was found only in one animal. In this case 22% of MCs were strongly RyR-positive. No staining was registered in 44-73% of MCs. Double staining with 5HT and these receptor markers proved that indeed only a part of MCs is actively secreting. Resting cells with only 5HT-immunopositivity are often visible. The activational state of MCs is changing at higher estrogen/testosterone level, thus with the secretion of neuromodulators they might alter sexual and parental behavior of the animals.

An isolation method for assessment of brain mitochondria function in neonatal mice with hypoxic-ischemic brain injury.

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 +/- 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.

Doppler cerebro-umbilical ratio and fetal biophysical profile in the assessment of peripartal cardiotocography in growth-retarded fetuses.

OBJECTIVE: The aim of the study was to examine the value of the fetal biophysical profile (FBP) and the Doppler cerebro-umbilical ratio (C/U) in the assessment of peripartal cardiotocography (CTG) in growth-retarded fetuses. METHODS: The prospective study included 58 pregnant women with singleton pregnancy from 28 to 42 weeks of gestation with clinically and ultrasonically verified late intrauterine growth retardation (IUGR). After assessment of the FBP, flow velocity waveforms from the umbilical and middle cerebral arteries were recorded and the C/U ratio was calculated. The C/U ratio and the FBP were assessed twice a week. The last peripartal CTG was used as an outcome parameter. RESULTS: The FBP score was statistically significant when associated with peripartal CTG (p < 0.001). The mean value of the FBP was 7.77 +/- 0.28 for infants with a normal peripartal CTG, 6.13 +/- 0.41 for infants with a prepathological CTG and 4.40 +/- 0.60 for infants with a pathological peripartal CTG. There was also a statistically significant association between the C/U ratio > or =1 and normal CTG (p < 0.005), but there was no statistically significant difference between prepathological and pathological CTG in relation to C/U ratio values (p > 0.05). Normal and pathological peripartal CTG was correlated with the perinatal outcome, but prepathological CTG was not correlated with results of the perinatal outcome (p > 0.05). CONCLUSIONS: Our results showed that both methods of fetal monitoring need to be used in perinatal monitoring of growth-retarded fetuses. The two methods can be used as important parameters in deciding to end pregnancies with IUGR when pathological values occur.

Assessment of retinal function and characterization of lysosomal storage body accumulation in the retinas and brains of Tibetan Terriers with ceroid-lipofuscinosis.

OBJECTIVE: To characterize lysosomal storage body accumulation in the retina and brain of Tibetan Terriers with ceroid-lipofuscinosis and determine whether the disease in these dogs is accompanied by impaired retinal function and retinal degeneration. ANIMALS: Three 7- to 10-year-old Tibetan Terriers with ceroid-lipofuscinosis and 1 healthy 5-year-old Tibetan Terrier. PROCEDURE: Owners completed a questionnaire to identify behavioral and physical signs indicative of ceroid-lipofuscinosis. Neurologic, behavioral, and ophthalmologic evaluations, including full-field electroretinograms, were performed on each dog. Fluorescence, light, and electron microscopy were performed on specimens of retina, cerebral cortex, and cerebellum of all dogs postmortem. RESULTS: Behavioral assessments of the affected dogs revealed moderate visual impairment in low-light conditions but good vision in bright light. On funduscopic evaluation of these dogs, abnormalities detected ranged from none to signs of moderately advanced retinal degeneration. Compared with findings in the control dog, electroretinography revealed depressed rod cell function with some impairment of cone cell function in the affected dogs. Morphologically, disease-specific storage bodies were detected in retinal Müller cells and neurons, particularly in ganglion cells, and in cells of the cerebral cortex and cerebellum in affected dogs. Substantial photoreceptor cell loss and disruption of photoreceptor outer segment morphology appeared to develop late in the disease. IMPLICATIONS FOR HUMAN MEDICINE: The similarities between ceroid-lipofuscinosis in Tibetan Terriers and some forms of ceroid-lipofuscinosis in humans suggest that the canine disease may have a genetic and biochemical basis similar to that of one of the ceroid-lipofuscinosis disorders in humans.

MR imaging assessment of myelination in the very preterm brain.

BACKGROUND AND PURPOSE: MR imaging was performed in very preterm infants by using an MR imager in the neonatal intensive care unit. The aims of this study were to assess the development of myelination in the preterm brain based on MR imaging findings and to compare the ability of T1-weighted conventional spin-echo, inversion recovery fast spin-echo, and T2-weighted fast spin-echo MR imaging to show myelination in these infants. METHODS: MR imaging was performed for 26 preterm infants with a median gestational age of 28 weeks who had normal neurodevelopmental outcomes at 2 years corrected age. RESULTS: Myelin was evident in the gracile and cuneate nuclei and fasciculi, vestibular nuclei, cerebellar vermis, inferior and superior cerebellar peduncles, dentate nucleus, medial longitudinal fasciculus, medial geniculate bodies, subthalamic nuclei, inferior olivary nuclei, ventrolateral nuclei of the thalamus, decussation of the superior cerebellar peduncles, medial lemnisci, lateral lemnisci, and inferior colliculi at < or = 28 weeks gestational age. From this gestational age, myelination was not visualized at any new site until 36 weeks gestational age, when myelin was visualized in the corona radiata, posterior limb of the internal capsule, corticospinal tracts of the precentral and postcentral gyri, and lateral geniculate bodies. T2-weighted fast spin-echo MR imaging showed myelin in gray matter nuclei at an earlier gestational age than did T1-weighted conventional spin-echo or inversion recovery fast spin-echo MR imaging. T1-weighted conventional spin-echo MR imaging showed myelin earlier in some white matter tracts in the preterm brain. CONCLUSION: Myelination was evident in numerous gray and white matter structures in the very preterm brain. A knowledge of myelination milestones will allow delays to be detected at an early stage.

Assessment of benzydamine N-oxidation mediated by flavin-containing monooxygenase in different regions of rat brain and liver using microdialysis.

N-Oxidation of benzydamine (BZY) mediated by flavin-containing monooxygenase (FMO) was evaluated by microdialysis in vivo in different regions of rat brain and liver. The probe was implanted into local regions of the brain, such as the olfactory bulb, cerebral cortex, corpus striatum, hippocampus and cerebellum, or the hepatic lobe. By perfusing BZY via the probe, BZY N-oxide was identified in the dialysate. The estimated concentrations of BZY N-oxide in extracellular fluid were almost the same as those in the olfactory bulb, hippocampus and cerebral cortex, half the concentration in the hepatic lobe; however, the concentration in the corpus striatum was lower and that in the cerebellum was higher than in the other regions. These results demonstrate that the extracellular concentration of BZY N-oxide formed in vivo was unexpectedly high in every brain region.

Subcellular localization of docosahexaenoic acid and arachidonic acid omega-hydroxylation activity in the brain, liver and colonic adenocarcinoma.

A homogenate of rat brain, rat liver or human colonic well differentiated adenocarcinoma was prepared in 250 mM sucrose isoosmolaric buffer (pH 7.6) and fractionated by differential centrifugation at 10(3), 10(4) and 10(5) g. Each precipitate or supernatant was incubated with NADPH and docosahexaenoic acid or arachidonic acid as a substrate for 30 min at 37 degrees c under aerobic conditions. omega-Hydroxydocosahexaenoic acid or omega-hydroxyeicosatetraenoic acid from an incubation mixture was detected by reversed-phase high-performance liquid chromatography-thermospray mass spectrometry with selected-ion monitoring. omega-Hydroxy polyunsaturated fatty acids were characterized by high intensity of the molecular ion (MH+) although common hydroxy polyunsaturated fatty acids were characterized by high intensity of the MH+ -H2O ion. For the rat brain, omega-hydroxylation activity (the amount of omega-hydroxy product produced in 30 min) was concentrated to a 10(3) g precipitate although the specific activity (the activity per 1 mg of protein) in the 10(3) g precipitate did not indicate superiority over other fractions. However, the specific activity of the rat brain increased on addition of a 10(4) or 10(5) g precipitate. For the rat liver, although omega-hydroxylation activity was concentrated to a 10(3) g precipitate, the specific activity was concentrated to a 10(5) g precipitate and the subcellular localization differed from that of rat brain. In the human colonic well differentiated adenocarcinoma, although omega-hydroxylation activity was relatively high in the 10(3) g supernatant, the specific activity was relatively high in the 10(3) g precipitates. These results suggest that there is a difference regarding subcellular localization of the omega-hydroxylation activity depending on the species of the organs.

Assessment of the distribution of mitochondrial ribosomal RNA in melas and in thrombotic cerebral infarcts by in situ hybridization.

In situ hybridization to mitochondrial ribosomal RNA (rRNA) has been used to study the distribution of mitochondria in paraffin-embedded autopsy brain tissue from two patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and other organs from one of the patients. Comparison of in situ hybridization and electron microscopic findings in an antemortem biopsy specimen of pylorus from the latter patient showed a close correspondence between the distribution of hybridization signal on light microscopy and of mitochondria in ultrathin sections. Strong hybridization signal was present over smooth muscle fibres of the muscularis externa, which contained abnormal accumulations of mitochondria on electron microscopy. Hybridization to sections of skeletal muscle confirmed previous reports of 'ragged-red' fibres in this disorder and of mitochondrial accumulations in the walls of intramuscular blood vessels. To try to elucidate the role of vessel wall accumulation of mitochondria in the genesis of the stroke-like lesions, the distribution of mitochondrial rRNA was assessed in sections of brain from both of the cases of MFLAS and several cases of atherothrombotic cerebrovascular disease. Blood vessels in and adjacent to the cerebral lesions of MELAS showed strong hybridization signal with the mitochondrial probes, as was also seen in infarcts of various ages in the control brains. Only weak signal was present in the walls of blood vessels distant from the lesions, in both MELAS and control brains. These findings suggest that mitochondria accumulate in vascular endothelium and tunica media as a normal response to cerebral infarction or ischaemia. The accumulation of mitochondria in the cerebral lesions of MELAS may, at least in part, be a reaction to the destructive effects of the underlying metabolic dysfunction.