Dense arrays of micro-needles for recording and electrical stimulation of neural activity in acute brain slices.

OBJECTIVE: This paper describes the design, microfabrication, electrical characterization and biological evaluation of a high-density micro-needle array. The array records from and electrically stimulates individual neurons simultaneously in acute slices of brain tissue. APPROACH: Acute slices, arguably the closest in-vitro model of the brain, have a damaged surface layer. Since electrophysiological recording methods rely heavily on electrode-cell proximity, this layer significantly attenuates the signal amplitude making the use of traditional planar electrodes unsuitable. To penetrate into the tissue, bypassing the tissue surface, and to record and stimulate neural activity in the healthy interior volume of the slice, an array of 61 micro-needles was fabricated. MAIN RESULTS: This device is shown to record extracellular action potentials from individual neurons in acute cortical slices with a signal to noise ratio of up to ∼15:1. Electrical stimulation of individual neurons is achieved with stimulation thresholds of 1.1-2.9 µA. SIGNIFICANCE: The novelty of this system is the combination of close needle spacing (60 µm), needle heights of up to 250 µm and small (5-10 µm diameter) electrodes allowing the recording of single unit activity. The array is coupled to a custom-designed readout system forming a powerful electrophysiological tool that permits two-way electrode-cell communication with populations of neurons in acute brain slices.

Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila.

In the fly visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. The cellular requirement for R1-R6 targeting was determined using loss-of-function mutations affecting different cell types in the lamina. nonstop (encoding a ubiquitin-specific protease) is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. We propose that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity.

brakeless is required for photoreceptor growth-cone targeting in Drosophila.

The R1-R6 subclass of photoreceptor neurons (R cells) in the Drosophila compound eye form specific connections with targets in the optic ganglia. In this paper, we report the identification of a gene, brakeless (bks), that is essential for R1-R6 growth cone targeting. In brakeless mutants, R1-R6 growth cones frequently fail to terminate migration in their normal target, the lamina, and instead project through it and terminate in the second optic ganglion, the medulla. Genetic mosaic analysis and transgene rescue experiments indicate that bks functions in R cells and not within the lamina target region. bks encodes a nuclear protein. We propose that it participates in a gene expression pathway regulating one or more growth cone components controlling R1-R6 targeting.

Practice compared. The UK and Australia.

OBJECTIVE: To compare the processes and cost of management of chronic health problems between a rural general practice in the UK and a similar practice in Australia. Patients were selected from three groups with either diabetes, asthma or depression. DESIGN: Case study, retrospective. This comparison was conducted during a 6 month practice exchange. SETTING: Wagin, Western Australia and Berwick upon Tweed, England. SUBJECTS: Ten people (five men, five women) were selected from each practice, for each disease type. Their records were culled and compared on the basis of how well they had satisfied the requirements of the protocols (where they existed) on the management of each disease. Other comparisons also included the number and cost of consultations, prescriptions and hospital stays. RESULTS: There was no apparent difference in the age/sex distributions of the populations studied. Comparisons of the three disease groups showed: Asthma-results similar on most counts, except hospital admissions. Protocol uniformly poorly followed. Diabetes-results similar, except use/cost of prescription drugs which was much greater in Australia. The UK was much better following its protocol. Depression-Australia again used significantly more prescription drugs. The use and cost of GP/specialist visits was higher overall in Australia. CONCLUSION: This study suggests that a rural UK practice provides more cost effective review of chronic health problems. Further study would be warranted to investigate the impression that this was achieved at the expense of patient and doctor satisfaction.

Cell cycle progression in the developing Drosophila eye: roughex encodes a novel protein required for the establishment of G1.

The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. These cells will then either commit to another round of cell division or differentiate into neurons. Although cell cycle synchronization occurs in roughex (rux) mutants, cells circumvent G1 and all cells enter S phase, including cells that would normally differentiate. This leads to defects in early steps of pattern formation and cell fate determination. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star). rux encodes a novel protein of 335 amino acids. We propose that rux functions as a negative regulator of G1 progression in the developing eye.

Comparative teratogenicity and metabolism of all-trans retinoic acid, all-trans retinoyl beta-glucose, and all-trans retinoyl beta-glucuronide in pregnant Sprague-Dawley rats.

When single large equimolar doses (0.38-0.41 mmol/kg BW) of all-trans retinoic acid (RA), all-trans retinoyl beta-glucose (RBGL), and all-trans retinoyl beta-glucuronide (RBG) are administered orally in oil on day 8.5 of pregnancy to Sprague-Dawley rats, RA and RBGL proved highly teratogenic, whereas RBG was not. Indeed, fetuses from RBG-treated dams were 16% heavier (P < 0.01) than control fetuses. After dosing with RA and RBGL, RA appeared in large amounts within 0.5 h in the maternal plasma and within 1.0 h in the embryo. In contrast, orally administered RBG seemed to be absorbed much more slowly, to be converted very slowly to RA, and not to accumulate either as RBG or as RA in the embryo. When incubated in vitro with embryos and attached membranes, however, both all-trans RBG and all-trans RA were partially converted to 13-cis RA. The nonteratogenicity of RBG, in contrast to RA, seems to be due to a much slower rate of GI absorption, a slow rate of hydrolysis to RA, a limited passage from the maternal circulation into the embryo, and a lower inherent toxicity.

Metabolism in vivo of all-trans-[11-3H]retinoic acid after an oral dose in rats. Characterization of retinoyl beta-glucuronide in the blood and other tissues.

Soon after [11-3H]retinoic acid (RA) (1.1 x 10(8) d.p.m.) was administered orally to rats either as a large dose (115 micrograms = 0.38 mumol/rat) or mixed with unlabelled RA as a huge dose (22 mg = 73.33 mumol/rat), retinoyl beta-glucuronide (RAG) was identified and characterized as a significant metabolite in the serum and small intestine. Of the administered dose, 70% remained unchanged as retinoic acid in the stomach up to 1 h. Significant amounts of 5,6-epoxyretinoic acid, 4-hydroxyretinoic acid, esters of retinoic acid and several polar retinoids, including 4-oxoretinoic acid, were also detected in the stomach. No significant difference was observed in the nature of the retinoids found after a large or a huge dose; however, the ratio of RAG/RA was higher after a huge dose than after a large dose. Thus RAG, which is biologically active in vivo and in vitro, is formed quickly in significant amounts in tissues after a dose of RA.

Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.

cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.

Metabolism, plasma transport and biliary excretion of radioactive vitamin A and its metabolites as a function of liver reserves of vitamin A in the rat.

Groups of 7-12 weanling Sprague-Dawley male rats were fed graded daily doses of vitamin A (5-176 micrograms retinol) for 7 or 12 weeks. Final mean liver concentrations of vitamin A, which ranged from 0.4 to 331 micrograms retinol per gram, depended both on the daily dose given and on the length of the feeding period. The mean serum retinol concentration was 24 micrograms/dl at the lowest liver vitamin A concentration, approached a plateau of 40 micrograms/dl at a liver concentration of 5-10 micrograms/g, and then very slowly increased with higher levels of vitamin A in the liver. Seven days after the oral administration of a standard dose (4.6 microCi) of 11,12-[3H2]retinyl acetate, during which period rats were fed the customary vitamin A-containing diet, bile was collected via bile duct cannulae for 1-4 hours, and then the livers and serum were extracted and analyzed. The key relationships defined were: 1) that the mean ratio of specific activities of retinol in serum to that in liver was 0.65 +/- 0.05 (SEM) (range: 0.46-0.81) at daily retinol intakes of 8-176 micrograms/day, 2) that the ratio did not vary systematically with vitamin A intake or liver reserves and 3) that the mean excretion rate of vitamin A metabolites in the bile was invariant at 0.28 microgram retinol metabolites per milliliter of bile up to a liver vitamin A concentration of 32 micrograms retinol per gram, but then increased rapidly by eightfold to a maximal rate of 2.4 micrograms retinol metabolites per milliliter of bile at a liver vitamin A value of 140 micrograms retinol per gram.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver concentrations of vitamin A and carotenoids, as a function of age and other parameters, of American children who died of various causes.

To assess the age-dependent vitamin A status of children, liver samples taken at autopsy from 170 American children 0-15 yr of age were analyzed for vitamin A and carotenoids. The median liver vitamin A concentration at birth was low (11 micrograms retinol/g), remained constant to 3 mo, rapidly increased to 4 yr (130 micrograms/g) and then remained constant into adolescence. In contrast the vitamin A status of premature infants deteriorated after birth. Of infants less than 3 mo, approximately one-fourth and two-thirds showed liver vitamin A concentrations less than or equal to 5 micrograms retinol/g and less than or equal to 20 micrograms/g, respectively. On the other hand, essentially all infants greater than or equal to 6 months showed an adequate vitamin A status, defined as liver stores greater than 20 micrograms retinol/g liver. Liver carotenoid concentrations did not meaningfully correlate with age or with vitamin A concentrations. Parameters that did not significantly affect the vitamin A concentration were: 1) height and weight in infants less than 1 mo, except in the highest weight-height groups, 2) sex, although values of females were slightly higher than males, and 3) causes of death.

The storage form of vitamin A in rat liver cells.

After collagenase treatment of perfused rat liver, isolated washed hepatocytes (parenchymal cells) and washed nonparenchymal cells contained 13-53% and 5-9%, respectively, of the total vitamin A in rat liver. After Pronase E and DNase digestion of liver, nonparenchymal cells contained 1-6% of the total liver vitamin A content. By density-gradient centrifugation, hepatocytes were divided into six fractions, which contained fewer lipid globules per cell and less vitamin A per cell as the cell density increased. In our procedures, lipocytes could not be isolated after either collagenase or pronase E and DNase digestion of liver. Of the total liver vitamin A, 40-80% was found in very low density, vitamin A-containing globules, which have a median diameter of 1.7 microns (range 0.4-4.6 microns), show intense yellow-green fluorescence under UV illumination and contain greater than 95% of their vitamin A in ester form. The distribution of vitamin A among cells and vitamin A globules in rats dosed with vitamin A was the same as in undosed rats. The possible interaction of lipocytes and different classes of hepatocytes in the storage and mobilization of vitamin A is discussed.

Variations in liver concentrations of iron and vitamin A as a function of age in young American children dying of the sudden infant death syndrome as well as of other causes.

Concentrations of total iron and of vitamin A were determined in the livers of 33 Midwestern children, varying in age from 2 weeks to 9 years, who had died from the sudden infant death syndrome (SIDS) as well as from other causes. The median liver iron concentration was high (500 micrograms/g wet weight) in infants up to one month of age, fell rapidly during the ensuing months, and reached a plateau of 125 micrograms/g at approximately 1 year of age. No differences were noted between male and female children. The median liver vitamin A concentration was very low (4 micrograms/g) in infants less than or equal to 1 month of age, rapidly increased during the ensuing months, and reached a value of 100 micrograms/g by one year. The ratio of vitamin A to iron in the liver increased roughly 100-fold during the first few years of life. Infants dying of SIDS had stores of iron and vitamin A that fell in the normal range for age. Only one subject of the 33 studied showed an iron value less than 50 micrograms/g, and no infants more than 5 months of age showed a vitamin A value less than 20 micrograms/g. Thus, the storage of iron and vitamin A appear to be normal in the livers of infants suffering from SIDS.