Pattern of polyamines and related monoacetyl derivatives in chick embryo retina during development

Polyamines and related monoacetyl derivatives were studied in chick embryo retina during development (6th–19th day). Putrescine, which is high in the first phase of retinogenesis, is necessary to sustain both tissue proliferation and via N-acetylputrescine, γ-aminobutyric acid synthesis. A later increase in spermidine and particularly spermine may play a role in the last phase of development when the retina reaches maturation. The presence of N-acetylspermidine already at the 8th day indicates that in chick embryo retina, putrescine synthesis can depend on two separate pathways. The first involves ornithine decarboxylase activity; the second, spermidine/spermine N¹-acetlytransferase and probably polyamine oxidase that converts spermidine to putrescine via N¹-acetylspermidine.     

[H]Spermine binding to synaptosomal membranes from the chick retina

The binding of [³H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [³H]spermine to synaptosomal membranes from plexiform layers of retina (P₁ and P₂) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (B_max=9.3 and 37 pmol/mg protein for P₁ and P₂, respectively). Kinetics of specific [³H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P₁ and P₂, respectively) with high affinity: K_app=61 and 67 nM for P₁ and P₂. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose–response curves for spermine, spermidine, and diethylene–triamine (DET) show different potencies for inhibiting [³H]spermine binding: spermine>spermidine>DET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

Acetylation of polyamines in mouse brain: subcellular and regional distribution

The acetylation of putrescine, cadaverine, spermidine, and spermine was examined in different subcellular fractions and regions of the mouse brain. Acetylation activity was confined to nuclear and microsomal fractions, which can acetylate all of these compounds. These fractions catalyze the formation of N8 but not N1-acetylspermidine. For the nuclear fraction the Km for putrescine was 3.5 mM; for cadaverine, 4.0 mM; for spermidine, 1.0 mM; and for spermine, 2.5 mM. The Vmax obtained were (pmol/mg protein/10 min): putrescine, 424; cadaverine, 705; spermidine, 239; and spermine, 467. The acetylation of spermidine was highest in the olfactory bulb and cerebellum. Putrescine and cadaverine acetylation were high in these areas, as well as in the midbrain. Spermine acetylation was rather uniform in all areas examined, except in the brain stem (pons-medulla) where enzyme activity was very low.     

Immunocytochemical localization of polyamines in the tiger salamander retina

The polyamines spermine and spermidine are present in neural tissue, but their functions there are not well understood. Recent work suggests that the NMDA subtype of glutamate receptors, other glutamate receptor subtypes, and certain K⁺-channels, are neural targets for polyamines. To better understand the neuron-specific roles of polyamines, we have developed antibodies that interact with spermine and spermidine in aldehyde-fixed tissue and used these antibodies in immunocytochemical studies to determine the cellular localization of these polyamines in the tiger salamander retina. The affinity-purified, polyclonal antibodies were highly specific for spermine and spermidine, exhibiting < 1 % cross reactivity with putrescine, and virtually no cross-reactivity with GABA, arginine, lysine, or glutaraldehyde. Polyamine labeling was most abundant in cells in the inner half of the inner nuclear layer and in the ganglion cell layer. Some cells in the outer half of the inner nuclear layer are labeled, and there was some labeling in both synaptic layers. Double-labeling experiments indicated (1) all GABAergic amacrine cells were polyamine-positive; and (2) all ganglion cells (identified by back-filling after microinjections of rhodamine in the optic nerve) were polyamine-positive. These results are consistent with a role for polyamines as modulators of NMDA receptor function and channel function in the inner retina.     

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration. J. Comp. Neurol. 387:148–156, 1997. © 1997 Wiley-Liss, Inc.     

Polyamine metabolism in different pathological states of the brain

Biosynthesis of the polyamines spermidine and spermine and their precursor putrescine is controlled by the activity of the two key enzymes ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (SAMDC). In the adult brain, polyamine synthesis is activated by a variety of physiological and pathological stimuli, resulting most prominently in an increase in ODC activity and putrescine levels. The sharp rise in putrescine levels observed following severe cellular stress is most probably the result of an increase in ODC activity and decrease in SAMDC activity or an activation of the interconversion of spermidine into putrescine via the enzymes spermidineN-acetyltransferase and polyamine oxidase. Spermidine and spermine levels are usually less affected by stress and are reduced in severely injured areas. Changes of polyamine synthesis and metabolism are most pronounced in those pathological conditions that induce cell injury, such as severe metabolic stress, exposure to neurotoxins or seizure. Putrescine levels correlate closely with the density of cell necrosis. Because of the close relationship between the extent of poststress changes in polyamine metabolism and density of cellular injury, it has been suggested that polyamines play a role in the manifestation of structural defects. Four different mechanisms of polyamine-dependent cell injury are plausible: (1) an overactivation of calcium fluxes and neurotransmitter release in areas with an overshoot in putrescine formation; (2) disturbances of the calcium homeostasis resulting from an impairment of the calcium buffering capacity of mitochondria in regions in which spermine levels are reduced; (3) an overactivation of the NMDA receptor complex caused by a release of polyamines into the extracellular space during ischemia or after ischemia and prolonged recirculation in the tissue surrounding severely damaged areas; (4) an overproduction of hydrogen peroxide resulting from an activation of the interconversion of spermidine into putrescine via the enzymes spermidineN-acetyltransferase and polyamine oxidase. Insofar as a sharp activation of polyamine synthesis is a common response to a variety of physiological and pathological stimuli, studying stressinduced changes in polyamine synthesis and metabolism may help to elucidate the molecular mechanisms involved in the development of cell injury induced by severe stress.     

Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Cytoskeletal specializations at the rod photoreceptor distal tip

We have examined microtubules and microtubule-like elements within the toad rod photoreceptor outer segment in order to define regional specializations of the photoreceptor cytoskeleton. "Ciliary" microtubules were localized within the rod outer segment (ROS) by using thin section electron microscopy, immunofluorescence, and rapid-freeze deep-etch microscopy. All three methods showed that ciliary microtubules stop short of the extreme ROS distal tip, although abundant microtubule-like structures distinct from the ciliary microtubules were found within the distal 10-15 microns of the ROS tip. These heretofore undescribed "distal ROS tubules" are clustered at the clefts or incisures of the disk membrane stack and resemble microtubules in overall size and shape, although they are not closely related antigenically to tubulin. The distal ROS tubules are more abundant in green rods than red rods and vary in number during the daily light/dark cycle. Quantitation of these tubules at two time points during the light/dark cycle suggests that there are three- to fourfold more tubules in the ROS tip one hour after light onset than one hour before light onset. Retinas prevented from normal disk membrane shedding by separation of the retina from the adjacent pigment epithelium, failed to develop increased numbers of tubules after light onset. This suggests that the newly described distal ROS tubules may modulate or be modulated by light-induced interactions between the photoreceptors and pigment epithelium, such as those that occur during the disk shedding phase of membrane turnover.     

Accumulation of exogenous polyamines in gerbil brain after ischemia

Regionally selective delayed neuronal degeneration is a characteristic sequel of cerebral ischemia. Recent evidence indicates that changes in brain polyamine metabolism may be critical for nerve cell survival after ischemia. Within hours after ischemia, intracellular putrescine levels are greatly increased and remain elevated for days, whereas only minor changes are noted in the levels of the polyamines spermine and spermidine. In contrast, the extracellular levels of all polyamines are low after ischemia. Injections of polyamines following ischemia, however, can protect neurons in the gerbil brain from delayed cell death, with spermine being the most potent of the polyamines. In the present study, therefore, we sought to determine if increased polyamine uptake occurs in the brain after ischemia. In the hippocampal slice preparation, temperature-dependent uptake was unique for spermine, but not for spermidine or putrescine. Uptake of [¹⁴C]spermine was transiently increased after ischemia, peaking at 150% of control by 12–13 h and subsiding by 24 h. Intravenous injections of [³H]spermidine resulted in a postischemic accumulation of this polyamine throughout the forebrain parenchyma. We conclude that:

1. Active cellular uptake of spermine is transiently increased early after ischemia;
2. A nonspecific accumulation of exogenous polyamines occurs early after ischemia probably owing to a compromised blood-brain barrier, and
3. The findings indicate that exogenous polyamines can exert their effect directly in the brain after ischemia.

     

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full‐field light of various intensities. Light‐evoked membrane potential changes (ΔV_m) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark‐adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage‐gated Ca²⁺ influx (Q^Ca) and consequent glutamate release. Stimulus‐evoked glutamate release was quantified by the increase in membrane capacitance (ΔC_m). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔV_m/Q^Ca ratio equally at a given light intensity and inhibition did not alter the overall relation between Q^Ca and ΔC_m. However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔC_m unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non‐linear relationship between Q^Ca and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.     

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy

To investigate the role(s) of protein‐tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1^?/?/Tpst2 ^?/?) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein‐tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.     

The distributions of photoreceptors and ganglion cells in the California ground squirrel,Spermophilus beecheyi

The topographical distributions of photoreceptors and ganglion cells of the California ground squirrel (Spermophilus beecheyi) were quantified in a light microscopic study. The central retina contains broad, horizontal streaks of high photoreceptor density (40–44,000/mm²) and high ganglion cell density (20–24,000/mm²). The isodensity contours of both cell types are elliptical and oriented along the nasal-temporal axis. There are roughtly fivefold decreases in both photoreceptor and ganglion cell densities with increasing eccentricity, the lowest densities being found in the superior retina. Large transitions in cell density and retinal thickness occur across the linear optic nerve head. Rod frequency increases with increasing eccentricity, from 5 to 7% in the central retina to 15 to 20% in the periphery. Roughly 10% of the cones possess wide, dark-staining ellipsoids. These cones are uniformly distributed across the retina which suggests that they may belong to a separate cone class, possibly blue-sensitive cones. The ganglion cell soma size distribution is unimodal, with the majority of somata being 25–50 μm². Large ganglion cells (somata > 100 μm²) are rare in the central retina, but their frequency increases with increasing eccentricity. No evidence for separate size classes of ganglion cells was found. The gradual decrement of photoreceptor density across the ground squirrel retina suggests that there are only relatively small changes in acuity across much of the animal's visual space compared with species possessing either a narrow visual streak or fovea or area centralis.     

Interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina

Both double-label and intracellular electrophysiological recording techniques were utilized to investigate the interactions between enkephalin and γ-aminobutyric acid in the larval tiger salamander retina.Double-label studies revealed that the vast majority (96%) 96%) of enkephalin-immunostained amacrine cells also exhibit high affinity uptake of [³H]γ-aminobutyric acid. Electrophysiological evidence demonstrated that morphine and γ-aminobutyric acid exert opposite effects on a population of On-Off ganglion cells. γ-Aminobutyric acid decreased the activity of these cells, while enkephalin increased their activity. These findings support the idea that opiate-mediated pathways inhibit GABAergic pathways in the vertebrate retina.     

Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves

Radioactive putrescine, spermidine or spermine was injected into the right eye of normal goldfish and fish in which both optic nerves had been crushed 18 days earlier. Fish were sacrificed 0.25–21 days after injection. Trichloroacetic acid-soluble and -insoluble material was extracted from the right retina and both tecta and assayed for radioactivity (significant differences between left and right tecta suggesting axonal transport). The nature of the radioactivity in the TCA-soluble fraction was determined on an amino acid analyzer.

Results indicate that putrescine is not axonally transported in intact goldfish optic nerves, but that during regeneration of the optic nerve large amounts of putrescine are axonally transported at rates similar to the fast component of protein transport. Spermidine appears to be axonally transported both in intact optic nerves and in regenerating optic nerves, and at an intermediate rate of transport; the amount of spermidine transported is significantly increased during regeneration. Spermine is also axonally transported in intact and regenerating nerves, at a rate similar to the rapid rate of protein transport. The amount of spermine transported appears to be slightly less in regenerating than in intact nerves during early stages of regeneration, but increases during later stages of nerve regeneration.

The results suggest that putrescine and spermidine may be preferentially transported during nerve regeneration, while spermine and spermidine are transported extensively in intact nerves.     

Retinal degeneration in the nervous mutant mouse. I. Light microscopic cytopathology and changes in the interphotoreceptor matrix

Nervous is an autosomal recessive mutation in mice (gene symbol, nr) that produces a progressive cerebellar and retinal degeneration. We have examined various cytopathological features of the photoreceptor degeneration by light microscopy. An increase in the number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) is first seen at postnatal day (P) 11. Between P13 and P19 there is a rapid loss of photoreceptors, with the ONL about 60% the thickness of littermate controls at P19. Between P19 and 2.5 months of age, photoreceptor cell loss is minimal, and there is a relatively slow loss of these cells between 3 and 7.5 months of age. At 7.5 months, the ONL consists of single row of nuclei, most of which are lost over the ensuing months, although a few photoreceptor nuclei persist at 17 months of age and older. Both rods and cones are lost at comparable rates for the first 2 months of life, but rods are somewhat preferentially lost at later ages. A very slight central-to-peripheral gradient of photoreceptor degeneration exists in the nr/nr retina, but no superior-inferior hemispheric differences are evident. The rate, spatiotemporal gradient, and hemispheric similarity in photoreceptor degeneration are the same in albino nr/nr mice reared either in cyclic light or in the dark, and in pigmented nr/nr mice. Autoradiographic analysis of rod outer segment renewal shows that outer segment membranes are synthesized in nervous homozygotes. Rhythmic outer segment disc shedding and phagocytosis by the retinal pigment epithelium occur at approximately normal rates in nr/nr mice. Histochemical and immunocytochemical study of the interphotoreceptor matrix (IPM) reveals the exclusion of stainable IPM from the outer segment zone by lamellar whorls of outer segment membrane, accumulation of stainable IPM in the basal region of the outer segment zone, and the absence of an intense band of stainable IPM at the apical surface of the retinal pigment epithelium. These changes in the IPM are similar to those seen in the Royal College of Surgeons rat. However, comparison of cytopathological changes in these two mutants reveals that the IPM defect probably is not the primary cause of photoreceptor cell death in nr/nr mice, and that similar phenotypic appearance does not necessarily signify similar pathological processes. © 1993 Wiley-Liss, Inc.     

Retinal patterning in the zebrafish mutant Cyclops

Determination of cell fate in the vertebrate retina has been shown to be largely independent of lineage. After cell fates are determined, retinal neurons become organized in a precise laminar pattern. The mechanisms for this patterning could involve morphogens distributed in gradients or, alternatively, direct cell-cell interactions. In the zebrafish mutant cyclops (cyc^b16), most embryos have two partial retinas joined in the ventral midline. This presents developing retinal cells near the midline with abnormal cellular environments, whereas laterally the pattern of developing cells is normal. We examined the consequences of this for patterning in the mutant's retina. We found that the retinas are joined in the midline at the apical surfaces of the photoreceptor layers. A laminar pattern emerges in the midline that preserves normal positional relationships between retinal cell types locally but is abnormal with respect to patterning over the entire retina. Lateral to the midline, retinal patterning appears normal. Metabolic labeling experiments showed that late rounds of DNA synthesis precede the emergence of the novel pattern in this midline region. We conclude that these observations in the cyclops mutant are compatible with mechanisms of pattern formation in the retina involving local cell interactions. J. Comp. Neurol. 381:449-460, 1997. © 1997 Wiley-Liss, Inc.     

Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.