Morphological changes in paraboloid glycogen of the accessory cone of the chick retina by exposure to light (author''s transl)

We studied five patients in whom severe thrombocytopenia developed during intermittent intravenous heparin treatment of arterial and venous thrombosis. Platelet aggregation was demonstrated when heparin (0.5 U per milliliter) was incubated with the patients' citrated platelet-rich plasma or with normal platelet-rich plasma in the presence of the patients' serum. Antiplatelet antibody was not detected in the patient globulin fractions prepared from serum collected within one week after heparin withdrawal by use of the platelet factor 3 availability technic. When the studies were repeated with modifications to detect heparin-dependent antiplatelet antibodies, positive results were obtained in four of five patients. The data suggest that a casual relation, mediated by an immune mechanism, existed between heparin therapy and thrombocytopenia, and that this syndrome may occur more often than has previously.     

Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4 h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of N-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs. Combined with previous data on the expression of neurotrophin messenger RNAs following similar injuries, our results support the hypothesis that increased levels of neurotrophins and their receptors could protect against neuronal damage following a brain insult.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.     

Melatonin and exposure to constant light/darkness affects ovarian follicular kinetics and estrous cycle in Indian desert gerbil Meriones hurrianae

Health care providers are becoming increasingly involved with the development of clinical pathways as they take on more of the responsibilities of quality management and resource use. Although clinical pathways that are developed at the national, regional, or specialty organization level provide a framework for reference, locally developed pathways tailor care to patients who are served within an agency or community. As multidisciplinary teams develop pathways for patients with back and spine disorders, they create tangible definitions of quality. Variance tracking systems used in conjunction with pathways provide feedback on patient progress and outcomes. These guidelines have significant potential for the future.     

Ontogenesis of lipids in chick embryo retina

PURPOSE: The effects of embryonic development on lipid composition in the retina were studied in 7, 11, 15, and 18-day-old chick embryos and newly hatched chicks. METHODS: The proportions of phospholipids, free and esterified cholesterol, diacylglycerides, and free fatty acids were determined using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to determine the fatty acid composition. RESULTS: The major phospholipid species were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin. Concentrations of the analyzed components have been related to the chronology of concrete stages of retinal development. The fatty acid composition of the total lipids, (n-6):(n-3) and saturated: unsaturated fatty acid ratios, and other parameters are reported. The proportions of total saturated and total monounsaturated fatty acids decreased very little from day 7 to hatching, whereas total polyunsaturated fatty acids nearly doubled over the same period. The increase in C18:2(n-6) from day 11 onwards was not followed by a similar increase in C20:4(n-6), hence the C20:4 to C18:2 ratio decreased with age. CONCLUSIONS: The cholesterol:phospholipid ratio decreased from day 7 to day 15 and increased from day 15 to hatching. High proportions of esterified cholesterol, very probably originating in the retinal pigment epithelium, were also recorded. Total saturated and monounsaturated fatty acids decreased, while polyunsaturated fatty acids increased during the period of initial retinal growth.     

Developmentally regulated spontaneous activity in the embryonic chick retina

Even before birth and the onset of sensory experience, neural activity plays an important role in shaping the vertebrate nervous system. In the embryonic chick visual system, activity in the retina before vision has been implicated in the refinement of retinotopic maps, the elimination of transient projections, and the survival of a full complement of neurons. In this study, we report the detection of a physiological substrate for these phenomena: waves of spontaneous activity in the ganglion cell layer of the embryonic chick retina. The activity is robust and highly patterned, taking the form of large amplitude, rhythmic, and wide-ranging waves of excitation that propagate across the retina. Activity waves are most prominent and organized between embryonic days 13-18, coinciding with the developmental period during which retinal axons refine their connections in their targets. The spatial and temporal features of the patterns observed are consistent with the role of activity patterns in shaping eye-specific projections and retinotopic maps but inconsistent with the hypothesis that they specify lamina-specific projections in the tectum. Antagonists of glutamatergic and glycinergic transmission and of gap junctional communication suppress spontaneous activity, whereas antagonists to GABAergic transmission potentiate it. Based on these results, we propose that spontaneous activity in the ganglion cells is regulated by chemical inputs from both bipolar and amacrine cells and by gap junctional coupling involving ganglion cells.     

Lipid mediators in the rat retina: light exposure and trauma elicit leukotriene B4 release in vitro

Light exposure not only elicits a visual response but may also alter functional and structural characteristics of the retina. Furthermore, light exposure can lead to reversible or irreversible lesions of photoreceptors and pigment epithelium. Previous studies in our laboratory have shown that light liberates arachidonic acid from retinal membrane phospholipids mainly by activating the phospholipase A2. In this study we show that light and trauma elicit the synthesis of leukotriene B4 in the isolated rat retina in vitro. Male albino rats were dark adapted for 36 h, isolated retinae were taken, incubated and exposed a) either to darkness or to 5,000 lux of cool white fluorescent light for 5, 10 or 15 min at 37 degrees C, b) either to darkness or to 5,000 lux of cool white fluorescent light for 15 min at 0 degrees C or c) either to darkness or to 5,000 lux of cool white fluorescent light for 15 min at 37 degrees C with a 5-lipoxygenase inhibitor (zileuton). Eicosanoids were extracted and leukotriene B4 levels were determined by radioimmunoassay. Removal of retinae and incubation in darkness caused a significant rise in leukotriene B4 levels with increasing incubation time. This rise was further augmented significantly after light exposure. The leukotriene B4 levels obtained when incubating the retinae either at 0 degree C or with the lipoxygenase inhibitor zileuton as well as the high specificity of the radioimmunoassay indicate that the light- and trauma-elicited synthesis of leukotriene B4 is mediated by activating the 5-lipoxygenase. Leukotriene B4 may be involved, at least in part, in the pathogenesis of retinal diseases including light damage. Curr. Eye Res. 14: 1001-1008, 1995.     

Conductances evoked by light in the ON-beta ganglion cell of cat retina

We have addressed the role of Ca2+ channels in mossy fiber synaptic transmission and long-term potentiation (LTP). Whereas the induction of mossy fiber LTP is entirely normal when synaptic transmission is blocked by the glutamate receptor antagonist kynurenate, LTP is blocked in the absence of extracellular Ca2+. These findings suggest that presynaptic Ca2+ entry is essential for mossy fiber LTP. Therefore, the role of different types of presynaptic Ca2+ channels in synaptic transmission and LTP was investigated. Mossy fiber responses were little affected by the L-type Ca2+ channel blocker nifedipine. They were blocked partially by omega-conotoxin-GVIA (N-type) and almost entirely by omega-agatoxin-IVA (P-type). None of these antagonists blocked mossy fiber LTP, nor was its expression associated with a change in sensitivity of synaptic transmission to either of the two toxins. These results, together with previous findings, suggest that the induction of mossy fiber LTP is critically dependent on the entry of Ca2+ into the presynaptic terminal to trigger a series of steps resulting in the long lasting enhancement of evoked glutamate release. Whereas P-type Ca2+ channels are of primary importance in mossy fiber synaptic transmission, both the induction and expression of mossy fiber LTP can occur in the absence of P-type (or N-type) Ca2+ channels.     

Calcium-induced spikes in cultured pigment epithelium of chick retina

Resting membrane potentials, electrical resistance, and coupling of chick retinal pigment epithelium cells in tissue culture have been measured with micropipette electrodes. Topical application of NaCl, KCl, MgCl2, and CaCl2 produce rapid, reversible changes in the membrane potentials of these cells. CaCl2 uniquely induces a slow hyperpolarizing wave that can lead to barrages of depolarizing action potentials or spikes and a slow, reversible vacuolization of the cells.     

Post-mortem changes in the rabbit retina. A study by light microscopy

The course of post-mortem changes in rabbit retina has been followed. Short post-mortem periods are accompanied by degenerative changes limited mainly to the visual cells and retinal pigment epithelium. Long post-mortem periods are associated with degenerative changes throughout the retina. Retinal tissue maintained at room temperature was less affected than that kept at body temperature (37degreesC). Post-mortem changes are similar to those observed following periods of pressure induced ischaemia and it is thought that the mechanical effects of pressure on retinal tissue are minimal at the level of resolution afforded by light microscopy.     

Contactin/F11 and tenascin-C co-expression in the chick retina correlates with formation of the synaptic plexiform layers

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.     

A fine structural and E-PTA study of photoreceptor synaptogenesis in the chick retina

Photoreceptor synaptogenesis in the embryonic and hatchling chick retina was studied with conventional EM techniques and ethanolic phosphotungstic acid (E-PTA). The photoreceptors line up between 11 and 13 embryonic days with their undifferentiated synaptic bases facing the outer plexiform layer (OPL). E-PTA staining at 11 embryonic days does not reveal any para-membranous specializations of the receptors but numerous stained punctae adhaerentes are observed in the OPL. At 13 embryonic days neurites of presumed bipolar and horizontal neurons are aligned parallel to the bases of the receptors and cytoplasmic protrusions of the receptors project between some of these neurites to form dyad appositions. An osmiophilic undercoating, which is not E-PTA positive at this time, is present on the cytoplasmic face of the receptor membrane in these apposition regions. Between 13 and 15 embryonic days the filopodial protrusions of the receptors continue to elongate further and become aligned with neurites in dyad and triad appositions. The osmiophilic undercoating now extends along the entire inner surface of the receptor pedicle protrusions and becomes E-PTA positive. Between 15 and 17 embryonic days focal aggregations of osmiophilic and E-PTA stained material appear along the membranes of the protrusions and there is some E-PTA staining of the postsynaptic densities and intervening cleft material. Between 17 and 21 embryonic days mature ribbon synapses are observed on the surfaces of the conical-shaped, receptor pedicles where the ribbons and their synaptic vesicles are associated with the dense aggregations (arciform densities), seen earlier as isolated focal aggregations, and the receptor undercoating is restricted to non-synaptic regions. E-PTA staining shows that ribbons are positively stained around their borders only and that they are contiguous with the intensely stained arciform densities. The cleft material and postsynaptic densities of some synapses first stain as V-shaped junctions and later as Y-shaped junctions. These observations suggest that ribbon synaptic junction formation begins with an alignment of pre- and postsynaptic membranes and the presence of the receptor presynaptic membrane undercoating, followed by the appearance of the presynaptic arciform densities and some staining of the cleft material and postsynaptic densities. These events are followed by the appearance of synaptic ribbons which are associated with the presynaptic arciform densities and by a further differentiation of the cleft material and postsynaptic densities.     

Neurotrophin-3 antibodies disrupt the normal development of the chick retina

When chick embryos are treated with a monoclonal antibody specifically blocking the activity of neurotrophin-3 (NT-3), the development of the retina is profoundly affected. Fewer axons are found in the optic nerve, and the retina shows abnormalities in all layers. Early during retinogenesis, the proportion of dividing cells is higher in NT-3-deprived embryos compared with age-matched controls and that of differentiated neurons is smaller. The NT-3 receptor trkC is expressed early by a majority of retinal cells, and NT-3 is present in the retina at the earliest stage studied. Initially, it is located mainly in the pigmented epithelium, with a shift toward the neural retina as development proceeds. Thus, NT-3 seems to be an essential intrinsic signal acting early in development to promote the differentiation and survival of many retinal neurons.     

Unusual responses to light and darkness of ocular melatonin in European sea bass

Regulation by light and darkness of melatonin rhythms in the plasma and eye of the European sea bass (Dicentrarchus labrax) was studied. During light-dark cycles, plasma and ocular melatonin exhibited day-night changes with higher levels at mid-dark and at mid-light, respectively. Circulating melatonin levels were low in constant light but high in constant darkness (DD); ocular melatonin levels showed the reverse pattern. Plasma melatonin exhibited circadian rhythm for 1 cycle but the rhythm was no longer apparent on day 2. There was no circadian rhythm in ocular melatonin. Acute light exposure in DD decreased plasma melatonin but increased ocular melatonin. These results suggest that circulating melatonin may be used as a signal for darkness but ocular melatonin is used as a signal for the light phase.     

Effects of continuous light and darkness on the eyes of the troglobitic salamander Typhlotriton spelaeus

Larval Typhlotriton spelaeus collected from five caves in Pulaski Co., Missouri, were kept as larvae or induced to transform in darkness or continuous fluorescent illumination. Larvae maintained in darkness for 215 and 279 days had smaller eyes, smaller rod inner and outer segments, and fewer metaphase figures in the germinative zone of the neural retina than comparable larvae maintained in light (258 lux). Except for visual cell size, differences were small and for each characteristic exceptions were observed. One larva kept in light showed early retinal degeneration comparable to that in transformed adults to T. spelaeus. All larvae exhibited optomotor behavior both before and after the experiment. Among animals induced to transform by L-thyroxin and maintained in darkness 111 to 366 days, visual cell and pigment epithelium degeneration was more extensive and more frequent than in animals kept for the same length of time in light (237-298 lux). In darkness the frequency of animals with retinal degeneration increased between 111 and 366 days. In light some animals exhibited pigment epithelium reduction with normal visual cells, and others had free, pigmented cells in the subretinal space. These effects were not comparable to degeneration in darkness. Eyelids covered the eyes of only a few animals in both light and dark treatments. The extent of eyelid encroachment over the eye greater in darkness than in light. Most animals exhibited optomotor responses after experiments, but responses of animals kept in darkness were impaired in comparison to those of animals kept in light.     

Heterogeneity among neuroepithelial cells in the chick retina revealed by immunostaining with monoclonal antibody PM1

Endothelin (ET) 1 is a powerful vasoconstrictor of coronary arteries and may play a role in coronary spasm, atherosclerosis, and myocardial infarction. Previous studies have demonstrated that intracoronary ET caused marked vasoconstriction of the coronary circulation; however, it remains unclear which ET receptor types are present and which of these receptors mediate this vasoconstriction. To characterize the ET receptors present in dog coronary arteries, competition binding assays with radiolabeled ET-1 using ET-1, ET-3, ETA receptor antagonist BQ-123, and sarafotoxin S6c were performed. Three binding sites were apparent in the left circumflex coronary artery: an ETA receptor, a high-affinity ETB receptor, and a lower-affinity ETB receptor. To investigate the in vivo effects of ETB receptor stimulation, intracoronary sarafotoxin S6c, a highly selective ETB agonist, was administered in anesthetized open-chest dogs in a constant-pressure coronary artery perfusion model. Sarafotoxin S6c doses of 0.1 and 0.3 microgram caused a transient pronounced decrease in coronary resistance. Doses of 1.0 and 3.0 micrograms caused marked decreases in coronary diameter and blood flow, as well as myocardial segmental shortening. These effects of sarafotoxin S6c were not inhibited by constant infusion of BQ-123. The present study demonstrates the presence of ETB receptors in the canine coronary circulation that can mediate both vasodilation and vasoconstriction. These findings have important implications for an understanding of the pathophysiological function of ET in the coronary vasculature and for the development of therapeutically effective ET antagonists.     

A quantitative analysis of cells in the ganglion cell layer of the chick retina

This study investigated the organization of cells in the ganglion cell layer (GCL) using Nissl staining, retrograde cell degeneration with axotomy of the optic nerve, and retrograde cell labeling by injections of horseradish peroxidase (HRP) into the optic nerve of chicks (posthatching day 1 and 8, P-1 and P-8). The total number of cells in the GCL was 6.1 x 10(6) (P-1) and 4.9 x 10(6) (P-8), and the cell density was 14,300 cells/mm2 (P-1) and 10,400 cells/ mm2 (P-8) on average. Two high-density areas, the central area (CA) and the dorsal area (DA), were observed in the central and dorsal retinas in both P-1 (22,000 cells/mm2 in CA, 19,000 cells/mm2 in DA) and P-8 chicks (19,000 cells/mm2 in CA, 12,800 cells/mm2 in DA). The cell densities in the temporal periphery (TP) and the nasal (NP) peripheral retinas were 7,800 cells/mm2 and 12,500 cells/mm2, respectively, in P-1 and 5,000 cells/ mm2 and 8,000 cells/mm2, respectively, in P-8 chicks. The cell density in the temporal periphery was 35% (P-8) lower than in the nasal periphery in both P-1 and P-8 chicks. Thirty percent (1.9 x 10(6) cells in P-1) of the total cells in the GCL were resistant to axotomy of the optic nerve. The distribution of the axotomy-resistant cells showed two high-density areas in the central and dorsal retinas, corresponding to the CA (5,800 cells/mm2) and the DA (3,200 cells/mm2). These cells also exhibited a center-peripheral increase (2,200 cells/mm2 in the TP) in P-1 chicks, but the high-density area was not found in the dorsal retina of P-8 chicks. From these data and the HRP study, the number of presumptive ganglion cells in P-8 chicks was estimated to be 4 x 10(6) (8,600 cells/mm2 on average), and the density in each area was 13,500 (CA), 10,200 (DA), and 4,300 (TP) cells/mm2. The peripheral/ center ratios of the density of ganglion cells were significantly different along the nasotemporal and dorsoventral axes. The density of ganglion cells decreased more rapidly toward the temporal periphery (TP/CA ratio: 0.47 in P-1 and 0.32 in P-8) than toward the nasal periphery (NP/CA ratio: 0.67 in P-1 and 0.52 in P-8). In contrast, there was no significant difference in the peripheral/center ratios between the dorsal retina (DP/CA ratio: 0.6 in P-1 and 0.56 in P-8) and ventral retina (VP/CA ratio: 0.58 in P-1 and 0.51 in P-8). A small peak in the density of the presumptive ganglion cells was detected in the dorsal retina of both P-1 chicks (10,800 cells/mm2) and P-8 chicks (10,200 cells/mm2). The HRP-labeled cells were small in the CA (M +/- SD: 35.7 +/- 9.1 microm2) and DA (40.0 +/- 11.3 microm2), and their sizes increased toward the periphery (63.4 +/- 29.7 microm2 in the TP) accompanied by a decrease in the cell density. However, the axotomy-resistant cells did not significantly increase in size toward the peripheral retina (12.2 +/- 2.2 microm2 in the CA, 15.2 +/- 3.2 microm2 in the DA, 15.1 +/- 3.8 microm2 in the TP). The characteristic distribution of ganglion cells could be related to visual behavior based upon the specialization of avian visual fields.     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Plasmids derived from bacteriophage lambda are known as lambda plasmids. These plasmids contain the ori lambda region and lambda replication genes O and P. Typical lambda plasmids also contain the cro gene, the product of which is a repressor of the pR promoter when present at relatively high concentrations. These genes stably maintain the plasmid in Escherichia coli at copy numbers of 20 to 50 per cell. According to a generally accepted model, stable maintenance of lambda plasmids is possible due to the Cro repressor autoregulatory loop (the cro gene is under control of pR). Here we demonstrate that lambda plasmids devoid of the Cro autoregulatory loop can also be stably maintained in E. coli strains. We present data for two such plasmids: pTC lambda 1 in which the pR-cro region has been replaced by the ptetA promoter and the tetR gene (coding for the TetR repressor), and a standard lambda plasmid with inactivated cro gene (lambda cro-null plasmid). Thus, the presence of the Cro repressor autoregulatory loop does not appear to be essential to the maintenance of lambda plasmids in vivo.