The dendritic architecture of the cholinergic plexus in the rabbit retina: Selective labeling by glycine accumulation in the presence of sarcosine

The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles. J. Comp. Neurol. 421:1-13, 2000. (C) 2000 Wiley-Liss, Inc.

The fountain amacrine cells of the rabbit retina

We have characterized a distinctive type of bistratified amacrine cell in the rabbit retina at both the single cell and population levels. These cells correspond to the fountain amacrine cells recently identified by MacNeil and Masland (1998). The fountain cells can be distinguished in superfused retinal wholemounts labeled with nuclear dyes, thus enabling them to be targeted for intracellular injection with Neurobiotin. This revealed that the primary dendrites ascend steeply to sublamina b of the inner plexiform layer, where they form an irregular arbor at the border of strata 4 and 5. These dendrites then give rise to multiple varicose processes that descend obliquely to sublamina a, where they form a more extensive arbor in stratum 1. The fountain amacrine cells show strong homologous tracer coupling when injected with Neurobiotin, and this has enabled us to map their density distribution across the retina and to examine the dendritic relationships between neighboring cells. The fountain amacrine cells range in density from 90 to 360 cells/mm(2) and they account for 1.5% of the amacrine cells in the rabbit retina. The thick tapering dendrites in sublamina b form highly territorial arbors that tile the retina with minimal overlap, whereas the thin varicose processes intermingle in sublamina a. The fountain cells are immunopositive for gamma-aminobutyric acid and immunonegative for glycine. We further propose that these cells are homologous to the substance P-immunoreactive (SP-IR) amacrine cells in the cat retina and that they may account for a subset of the SP-IR amacrine cells in the rabbit retina.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 mu M inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited similar to 80% by 5 mu M LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited similar to 70% by 0.3 mu M and 5 mu M LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality. (C) 1997 Academic Press Limited.

Seven retinal specializations in the tubular eye of the deep-sea pearleye, Scopelarchus michaelsarsi: A case study in visual optimization

The deep-sea pearleye, Scopelarchus michaelsarsi (Scopelarchidae) is a mesopelagic teleost with asymmetric or tubular eyes. The main retina subtends a large dorsal binocular field, while the accessory retina subtends a restricted monocular field of lateral visual space. Ocular specializations to increase the lateral visual field include an oblique pupil and a corneal lens pad. A detailed morphological and topographic study of the photoreceptors and retinal ganglion cells reveals seven specializations: a centronasal region of the main retina with ungrouped rod-like photoreceptors overlying a retinal tapetum; a region of high ganglion cell density (area centralis of 56.1x10(3) cells per mm(2)) in the centrolateral region of the main retina; a centrotemporal region of the main retina with grouped rod-like photoreceptors; a region (area giganto cellularis) of large (32.2+/-5.6 mu m(2)), alpha-like ganglion cells arranged in a regular array (nearest neighbour distance 53.5+/-9.3 mu m with a conformity ratio of 5.8) in the temporal main retina; an accessory retina with grouped rod-like photoreceptors; a nasotemporal band of a mixture of rod-and cone-like photoreceptors restricted to the ventral accessory retina; and a retinal diverticulum comprised of a ventral region of differentiated accessory retina located medial to the optic nerve head. Retrograde labelling from the optic nerve with DiI shows that approximately 14% of the cells in the ganglion cell layer of the main retina are displaced amacrine cells at 1.5 mm eccentricity. Cryosectioning of the tubular eye confirms Matthiessen's ratio (2.59), and calculations of the spatial resolving power suggests that the function of the area centralis (7.4 cycles per degree/8.1 minutes of are) and the cohort of temporal alpha-like ganglion cells (0.85 cycles per degree/70.6 minutes of are) in the main retina may be different. Low summation ratios in these various retinal zones suggests that each zone may mediate distinct visual tasks in a certain region of the visual field by optimizing sensitivity and/or resolving power.

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.

Improved beef production from supplementation of Hereford, Brahman and crossbred cattle grazing low and medium quality pastures in the subtropics of Australia

Subtropical grasslands are low in organic matter digestibility (OMD) (0.60) and nitrogen (N) (15 g/kg) for much of the year and this limits cattle production which is characterized by low calving rates and low weaning weights. Production has been based on Bos taurus British breeds of cattle but this is changing and now many breeding herds comprise B, indicus cows and their crosses. This change has increased some aspects of production, but low calving rates persist. A 4-year study was undertaken with a view to improve calving rates and weaner output by supplementing cows grazing either native or improved pastures with a high protein oilseed meal (cottonseed meal; CSM) on four sites. These sites were subdivided into a total of 36 paddocks to allow for two replications in a 3 breeds X 3 supplementation rates X 2 pastures factorial design. Selected cows (no. = 216) from Hereford (H), Brahman (B) and Brahman X Hereford (BH) breed types were set to graze either native pastures (0.45 to 0.62 OMD, 8 to 15 g N per kg; low quality) or improved pastures (0.47 to 0.67 OA ID, 10 to 22 g N per kg; medium quality). Cows were given either 0, 750 or 1500 g/day of CSM for 130 days from calving until 4 weeks into a 12- to 13-week mating period. The CSM was given as two meals per week. Live weight at mating of cows on the low quality pasture was increased (P < 0.01) over those not supplemented by feeding either 750 g CSM per day (H and B cows) or 1500 g CSM per day tall cows). There was no significant effect of supplementation on the mating weights of B cows grazing the medium quality sites. Calving rate of B cows was not increased by their supplementation on either low (4-year mean 58.3 %) or medium quality pastures (66.8%) but did tend to be higher in H cows when supplemented at 1500 g CSM per day on the low (66.7 v. 78.0 (s.e. 6.09) %; P < 0.1) and medium quality pastures (70.5 v. 93.5 (s.e. 4.72) %). An increased calving rate (65.8 (s.e. 6.6) % to 83.2 (s.e. 5.82) % in supplemented BH cows grazing low quality pastures approached significance (P < 0.1) when given CSM at 1500 g/day but there was no increased trend in calving rate when this breed type was supplemented on medium quality pastures. Weaning weights of calves from and B and BH cows were increased (P < 0.05) by supplementation of their darns at 750 g/day and for calves weaned from H cows supplemented at 1500 g/day of CSM. Supplementation at 1500 g/day on low quality pastures increased weaner output per cow mated by 120% for H, by 65% for BH cows and by 50% for B cows. Weaner output was increased by 34 and 40%, respectively, for B and H cows when supplemented at 750 g/day and grazing medium quality pastures but there teas no significant effect of supplementation on output from BH cows. Responses in many parameters differed between years. These results were interpreted as a response to the protein in the oilseed meal supplement by B, taurus and B. taurus X B. indicus cross cows grazing on the subtropical pastures. The study also highlighted that responses to the meal differed between breed types, between the quality of the grazed pasture and between the years of supplementation.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

Relationships between blood pressure and heart rate in the saltwater crocodile Crocodylus porosus

The cardiac limb of the baroreflex loop was studied in the saltwater crocodile Crocodylus porosus, The classical pharmacological methodology using phenylephrine and sodium nitroprusside was used to trigger blood pressure changes, and the resulting alterations in heart rate were analysed quantitatively using a logistic function. Interindividual differences in resting heart rates and blood pressures were observed, but all seven animals displayed clear baroreflex responses. Atropine and sotalol greatly attenuated the response. A maximal baroreflex gain of 7.2 beats min(-1) kPa(-1) was found at a mean aortic pressure of 6.1 kPa, indicating the active role of the baroreflex in a wide pressure range encompassing hypotensive and hypertensive states. At the lowest mean aortic pressures (5.0 kPa), the synergistic role of the pulmonary-to-systemic shunt in buffering the blood pressure drop also contributes to blood pressure regulation, Pulse pressure showed a better correlation,vith heart rate and also a higher gain than mean aortic, systolic or diastolic pressures, and this is taken as an indicator of the existence of a differential control element working simultaneously with a linear proportional element.