Amino acid signatures in the detached cat retina

PURPOSE: Expressions of certain macromolecules are altered by experimental retinal detachment in the cat. Related alterations in micromolecular signatures of neurons, Müller cells, and the retinal pigment epithelium (RPE) were investigated. METHODS: High-performance immunochemical mapping, image registration, and quantitative pattern recognition were combined to analyze the amino acid contents of virtually all retinal cell types after 3 to 84 days of detachment. RESULTS: Retinal micromolecular signatures showed a spectrum of alterations. The glutamate contents of Müller cells increased and remained elevated for weeks after detachment. Multispectral signatures of Müller cells showed massive metabolic instability in early detachment stages that ultimately resolved as a homogeneous profile significantly depleted in glutamine. Retinal pigment epithelial cell signals also changed dramatically, displaying an initial glutamate spike and then a prolonged decline, even as taurine levels followed an opposite pattern of initial loss and slow restoration. Neurotransmitter signatures of surviving neurons showed extensive precursor-level variation, and, in one case, GABAergic horizontal cells displayed anomalous sprouting. CONCLUSIONS: Dramatic changes in Müller cell amino acid signatures triggered by retinal detachment are partially consistent with losses in glutamine synthetase activity. Taurine signal variations suggest that orthotopic RPE cells attempt to regulate abnormal taurine concentrations in the enlarged subretinal space. Surviving neurons possess characteristic neurotransmitter signals, but their metabolite regulation seems abnormal. On balance, microchemical and structural anomalies develop in the detached cat retina that represent serious barriers to recovery of normal visual function.     

ON-OFF amacrine cells in cat retina

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b) but extends some dendrites to sublamina a. Both of these cell types have axon-like processes that extend > 800 microns from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 +/- 0.3 mm diameter) that were broader than their dendritic arbors (425 +/- 35 microns diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-alpha cell.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.     

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.     

Amino acid metabolism in liver tissue

Following determination by the method of ion-exchange chromatography of free amino acids in the blood drawn from the portal and liver veins and the abdominal aorta 30 minutes after introduction of a casein suspension to rats it was found that only 50 per cent of amino acids with ramified chain are retained in the liver. Most of the other amino acids, however, become metabolized largely in the liver and it is only their insignificant part that goes into the general circulation. These data may be of use in considering problems related to the peculiarities of metabolism of amino acids with ramified chain.     

Neurochemical architecture of the normal and degenerating rat retina

We used post-embedding immunocytochemistry to determine the cellular localization of glutamate, gamma-amino butyric acid (GABA), glycine, aspartate, glutamine, arginine, and taurine in the normal and degenerating rat retina. Müller's cell function was also evaluated by determining the uptake and degradation characteristics for glutamate. Immunocytochemical localization of amino acids in adult Royal College of Surgeons (RCS) and control rat retinas were similar with respect to cell classes. Differences in the intensity of labelling for glutamate, aspartate, glutamine, and glycine were observed in several classes of neurons, but the most prominent differences were shown by bipolar cells of the adult RCS rat retina. In addition, glutamine labelling within Müller's cells was higher in the RCS rat than the control. These changes may have occurred because of alterations in the glutamate production or degradation pathways. We tested this hypothesis by determining Müller's cells glutamate uptake and degradation characteristics in adult and postnatal day 16 RCS retinas. High affinity uptake of 3[H]-glutamate revealed an accumulation of grains over Müller's cell bodies in the adult RCS retina implying glutamate degradation anomalies. We confirmed anomalies in glutamate metabolism in RCS Müller's cells by showing that exogenously applied glutamate was degraded over a longer time course in postnatal day 16 RCS retinas, compared to control retinas. Differences in arginine immunoreactivity in adult and immature RCS retinas conform to the presumed dysfunction of Müller's cells in these degenerating retinas. The anomalies of amino acid localization, uptake and degradation lead us to conclude that Müller's cells in the RCS retina show abnormal function by postnatal day 16; an earlier time to previously reported anatomical and functional changes in this animal model of retinal degeneration.     

Amino acid kinetics during hemodialysis

The kinetic of essential amino acids as well as of histidine and alanine in bilateral nephrectomized rabbits was investigated during a 3 hours hemodialysis. Dialysis, elimination and incorporation rates into plasma proteins were determined for all applied amino acids. Total elimination rates of all the investigated amino acids varied. From 5.31% (leucine) to 75.51% (alanine) of the injected labelled amino acids were eliminated in the dialysate. There was an exponential decrease in the dialysis of essential amino acids and histidine during the period of investigation due to the high incorporation rate into plasma proteins. The kinetic of alanine was different due to a slow incorporation rate leading to a higher elimination rate. The fast incorporation of intravenously applied essential amino acids and histidine during dialysis demonstrates that the existing protein deficiency in renal failure can be influenced positively by infusion of amino acids.     

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.     

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.     

Effects of intravenous iodoacetate and iodate on pH outside rod photoreceptors in the cat retina

PURPOSE: Effects of intravenous iodoacetate (a glycolysis inhibitor) and iodate (a metabolism inhibitor selective to retinal pigment epithelium) on light-evoked alkalinizations and hypoxia-induced acidifications were studied in the dark-adapted cat retina, in vivo, to learn about pH regulation. METHODS: pH was recorded in the extracellular space surrounding rod photoreceptors with double-barrelled H(+)-selective microelectrodes. RESULTS: Intravenous infusion of 5 mg/kg iodoacetate-induced alkalinizations in the outer nuclear layer and suppressed both light-evoked and hypoxia-induced pH responses immediately. Iodate injection (30 mg/kg) produced acidifications in the subretinal space and affected light-evoked alkalinizations gradually but not hypoxia-induced acidifications. CONCLUSIONS: These results suggest that rods glycolysis plays an important role in both light-evoked and hypoxia-induced pH responses. And the retinal pigment epithelium may have little concern with light-evoked alkalinizations except that it plays an important role in regenerating the rhodopsin to be needed for the light responses of photoreceptors. Furthermore, the finding of the intravenous-iodoacetate-induced alkalinization in the outer nuclear layer supports that acid production by rods in the dark is originated from glycolysis to support the dark current. The iodate-induced acidification in the subretinal space indicators that the retinal pigment epithelium might actively transport acids from the subretinal space to the choroid.     

Amino acid metabolism and the energetics of growth

The nonessential amino acids are involved in a large number of functions that are not directly associated with protein synthesis. Recent studies using a combination of transorgan balance and stable isotopic tracers have demonstrated that a substantial portion of the extra-splanchnic flux of glutamate, glutamine, glycine and cysteine derives from tissue synthesis. A key amino acid in this respect is glutamic acid. Little glutamic acid of dietary origin escapes metabolism in the small intestinal mucosa. Furthermore, because glutamic acid is the only amino acid that can be synthesized by mammals by reductive amination of a ketoacid, it is the ultimate nitrogen donor for the synthesis of other nonessential amino acids. Because the synthesis of glutamic acid and its product glutamine involve the expenditure of adenosine triphosphate (ATP), it seems possible that nonessential amino acid synthesis might have a significant bearing on the energetics of protein synthesis and, hence, of protein deposition. This paper discusses the topic of the energy cost of protein deposition, considers the metabolic physiology of amino acid oxidation and nonessential amino acid synthesis, and attempts to combine the information to speculate on the overall impact of amino acid metabolism on the energy exchanges of animals.     

Amino acid metabolism of myeloma cells in culture

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 100% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.     

Scanning electron microscopy of normal and lased rabbit retina

This study presents the topography as seen by scanning electron microscopy of the rabbit retina in general and the photoreceptors in particular; and of large laser lesions in the retina.     

Synchronization of visual responses between the cortex, lateral geniculate nucleus, and retina in the anesthetized cat

Synchronization of spatially distributed responses in the cortex is often associated with periodic activity. Recently, synchronous oscillatory patterning was described for visual responses in retinal ganglion cells that is reliably transmitted by the lateral geniculate nucleus (LGN), raising the question of whether oscillatory inputs contribute to synchronous oscillatory responses in the cortex. We have made simultaneous multi-unit recordings from visual areas 17 and 18 as well as the LGN and the retina to examine the interactions between subcortical and cortical synchronization mechanisms. Strong correlations of oscillatory responses were observed between retina, LGN, and cortex, indicating that cortical neurons can become synchronized by oscillatory activity relayed through the LGN. This feedforward synchronization occurred with oscillation frequencies in the range of 60-120 Hz and was most pronounced for responses to stationary flashed stimuli and more frequent for cells in area 18 than in area 17. In response to moving stimuli, by contrast, subcortical and cortical oscillations dissociated, proving the existence of independent subcortical and cortical mechanisms. Subcortical oscillations maintained their high frequencies but became transient. Cortical oscillations were now dominated by a cortical synchronizing mechanism operating in the 30-60 Hz frequency range. When the cortical mechanism dominated, LGN responses could become phase-locked to the cortical oscillations via corticothalamic feedback. In summary, synchronization of cortical responses can result from two independent but interacting mechanisms. First, a transient feedforward synchronization to high-frequency retinal oscillations, and second, an intracortical mechanism, which operates in a lower frequency range and induces more sustained synchronization.     

Scanning electron microscopic observations of normal and regenerating nerve roots in the cat

The surface morphology of normal and regenerated nerve roots was studied using correlated scanning and transmission electron microscopic methods. Nerve roots of the cauda equina were either cut and rejoined or crossed from a segment above to a segment below. Good regeneration was observed in both experimental procedures. The regenerated nerve root sheath had alterations in surface structure created by extensive growth of collagen. Despite this collagen formation, regenerated axons crossed the anastomotic site with relative ease. Surface features of the regenerated axons were similar in appearance to those of the normal axon. Schwann cells were easily recognized, as were the collagen fibers of the endoneurium, although the endoneurium was more prominent and occupied more of the interaxonal space. Macrophages were identified as round structures with a laminated surface or as a honeycomb structure. Internal features of the regenerating axons were more difficult to identify, but mitochondria and a fibrous network were observed. These studies have demonstrated the application of scanning electron microscopic methods to visualize surface structures and cells in regenerated nerve roots.     

Amino acid uptake by fetal kidney slices

Kidney slices of 7-17-week-old human fetuses were incubated for 90 min or less in Krebs-Ringer solution containing aminoisobutyric acid (AIB). Cell to medium ratios of AIB decrease with increasing fetal age. Uptake rate is slower than for mature human tissue. Both water content and extracellular space is high in embryonic tissue as compared to adult.