Biomedical subjects

P Witkovsky

Publications and source records attributed to P Witkovsky.

At least 55 records · Page 3Linked to original sources

PubMed1987

Morphological changes induced in turtle retinal neurons by exposure to 6-hydroxydopamine and 5,6-dihydroxytryptamine.

Following intraocular injection of the dopamine neurotoxin 6-hydroxydopamine (10-50 micrograms on two successive days in a Ringer vehicle containing ascorbate and pargyline) and an incubation period of 1 to 18 days, degeneration was noted in presumptive amacrine cells in the retina of the turtle, Pseudemys scripta elegans. Injection of vehicle alone produced no effect. Affected perikarya initially showed swollen mitochondria, lysosomes and distended cisternae. At later stages the cells took on a darkened appearance. In contrast, affected amacrine processes in the inner plexiform layer became markedly distended and lost their cytoplasmic contents, resulting in empty, very swollen profiles. No degeneration was noted distal to the affected cell bodies, i.e. the affected cells were not interplexiform neurons. Cells lesioned by 6-hydroxydopamine were shown to accumulate [3H]dopamine. Intraocular administration of 5,6-dihydroxytryptamine (a single dose of 10-40 micrograms in the same vehicle) followed by 4-6 days incubation resulted in a marked darkening of certain bipolar cell axon terminals, cell bodies and Landolt's clubs. The toxic effects of 5,6-dihydroxytryptamine were blocked by zimelidine, a serotonin uptake blocker. Thus, these two neurotoxins have different targets in the turtle retina. At the highest dose tested, however, 6-hydroxydopamine did produce degenerative changes in the presumed serotonergic bipolar cell.

5,6-Dihydroxytryptamine↗

PubMed1987

Evidence for gluconeogenesis in the amphibian retina.

Evidence for the existence of a gluconeogenic pathway was provided in the amphibian retina. It was found that [3H]glutamate was converted to [3H]glucose derived from [3H]glutamate was incorporated into glycogen. The rate for this incorporation was found to be essentially the same in both light- and dark-adapted retinas: 0.147 vs. 0.142 nmol (mg protein X 2 hr)-1, respectively. However, the rate of incorporation was found to decline progressively with time. The rate for the incorporation of label derived from glutamate into glycogen was found to be considerably less than that for [3H]glucose: 10.2 nmol (mg protein X 2 hr)-1. The activity of a key gluconeogenic enzyme, fructose-1,6-bisphosphatase, also was demonstrated in retinal supernatants, approximately 1 nmol (mg X min)-1, and the activity of this enzyme was found to be inhibited both by adenosine monophosphate and by fructose-2,6-bisphosphate.

Adaptation, Ocular↗

PubMed1987

Dopaminergic mechanisms underlying the reduction of electrical coupling between horizontal cells of the turtle retina induced by d-amphetamine, bicuculline, and veratridine.

Previous studies have shown that dopamine, bicuculline, or d-amphetamine reduce the electrical and dye-coupling between the axon terminals of the horizontal cells of the turtle retina (see Piccolino et al., 1984). In the present study we observed similar effects following the application of veratridine. The actions of all these drugs were prevented by dopamine antagonists acting on D1 receptors such as flupenthixol and SCH 23390. However, in contrast to dopamine, the actions of d-amphetamine, bicuculline, and veratridine were attenuated or abolished by pharmacological agents (such as 6-OH-dopamine, alpha-methyl-p-tyrosine, or reserpine) known to reduce the release of dopamine from dopaminergic neurons. Moreover, the actions of veratridine and bicuculline were prevented by tetrodotoxin, indicating that one or more neurons in the dopamine pathway are spike-generating. We conclude that d-amphetamine, bicuculline, and veratridine reduce electrical coupling between the axon terminals of the turtle horizontal cells by promoting the release of endogenous dopamine from the dopaminergic amacrine cells previously identified (Witkovsky et al., 1984). Electron-microscopic observations revealed that 6-OH-dopamine selectively attacked this population of amacrine cells. No degenerating terminals were found adjacent to the horizontal cell axon terminals. On this basis, we postulate that dopamine reaches the horizontal cell by diffusion through the extracellular space.

Animals↗

PubMed1987

Center-surround organization of Xenopus horizontal cells and its modification by gamma-aminobutyric acid and strontium.

The properties of axon-bearing horizontal cells were studied by intracellular recording in the light-adapted Xenopus retina. In this adaptational state, the only receptor input was provided by red-sensitive cones. Measurements of response amplitude as a function of stimulus diameter indicated that different units were divisible into two compartments on the basis of their receptive field dimensions: one with a mean length constant, lambda, of 170 microns, the other with lambda = 450 microns. Lucifer Yellow dye injection revealed that units having small receptive fields invariably were cell bodies, whereas units having large receptive fields always were axons. For both horizontal cell bodies and axons, the waveform in response to large spots or annuli consisted of a hyperpolarizing on-transient, followed by a depolarizing rollback to a sustained plateau during light on, and a rapidly depolarizing off-transient that overshot the dark potential level. In contrast, in responses to small spots, the depolarizing rollback was much reduced or absent. However, for small field compartments only, when concentric annular stimuli were flashed around steadily illuminated, small, centered spots, the horizontal cell waveform consisted primarily of a depolarizing potential during light on. This depolarizing component was reduced selectively when the preparation was superfused with Ringer containing 5 mM gamma-aminobutyric acid (GABA). Following a brief exposure to 5-10 mM Sr2+, enhanced oscillatory potentials appeared in the horizontal cell light response during the depolarizing rollback and at light off. The modifications of the depolarizing rollback evoked by Sr2+ were antagonized by GABA. The strontium-induced alterations of the light response were not altered by 6 microM tetrodotoxin (TTX). The results are interpreted in terms of a feedback synaptic action exerted by horizontal cells upon cones, which is expressed as the depolarizing rollback component of the horizontal cell light-evoked waveform. The spatial properties of this component suggest that horizontal cell axons but not horizontal cell perikarya are capable of evoking a feedback signal in the cone. The actions of strontium indicate that calcium currents may play a role in shaping the feedback response. The ability of GABA to antagonize the Sr2+ effect upon the depolarizing rollback indicates that GABA may play a role in feedback.

Animals↗

PubMed1987

GABA and glycine modify the balance of rod and cone inputs to horizontal cells in the Xenopus retina.

Under mesopic conditions, the light-evoked waveform of horizontal cells in Xenopus retina reflects synaptic input from both rod and cone photoreceptors. These inputs interact non-linearly: the response to a weak red (cone-effective) flash is increased up to four-fold when the red stimulus falls on a green (rod-effective) background by reference to the response elicited by the same red flash on a dark field. We refer to this phenomenon as enhancement. It was not observed either when stimulus and field wavelengths were reversed or were of the same color. Enhancement was not altered by polarization of the horizontal cell membrane up to +/- 30 mV with extrinsic current. Enhancement could not be elicited with any combination of test and background wavelengths under photopic conditions. Superfusion of the retina with GABA resulted in a hyperpolarization of the horizontal cell membrane and an emphasis of the rod input to the horizontal cell light-evoked response. Picrotoxin depolarized the cell and favored the cone input. Enhancement was altered in predictable ways by these drugs. Glycine reduced selectively the cone input to the horizontal cell, whereas its antagonist, strychnine, increased the rod input. The results are interpreted to indicate that the excitability of the horizontal cell is modified both by distal and proximal retinal circuits; a glycinergic interplexiform cell probably plays a role in the latter pathway.

Animals↗

PubMed1986

Retinal neurochemistry of three elasmobranch species: an immunohistochemical approach.

We surveyed retinas of Raja erinacea, Mustelus canis, and Squalus acanthias for neurotransmitter substances by using antisera directed against the substances themselves or against their synthesizing enzymes. Both the peroxidase-antiperoxidase (PAP) and indirect fluorescent techniques were employed to visualize the primary antisera. In all three species positive results were obtained with antisera directed against tyrosine hydroxylase (TOH), glutamic acid decarboxylase (GAD), serotonin (5-HT), and leucine enkephalin (Lenk). Antisera directed against glucagon, neurotensin, beta-endorphin, vasoactive intestinal peptide, or bombesin failed to show any specific staining. Immunoreactivity was located in amacrine, interplexiform, and horizontal cells as well as in axons of the optic fiber layer. The four antisera labelled different amacrine cell classes, distinguished on the bases of perikaryal morphology and the distribution of cell processes in the inner plexiform layer (IPL). Amacrine cells that labelled with the same marker were seen to have different morphologies in the species studied. Thus, TOH-like immunoreactivity was distributed in layers 1, 3, and 5 of the IPL in Mustelus but only in layers 1 and 3 in Raja retina. GAD-like immunoreactivity was found diffusely over all layers of the IPL in Raja, but in Mustelus it was confined primarily to layers 1, 3, and 5 of the IPL. Lenk- and 5-HT-like immunoreactivities showed similar species variations. Two neurochemical classes of interplexiform cell were identified in this study. In Mustelus GAD-like and Lenk-like immunoreactive interplexiform cells were seen whereas in Raja only GAD-positive interplexiform cells were detected. In squalus no unequivocal demonstration of any interplexiform cell was made with these antisera. The GAD antiserum also labelled a subset of the horizontal cells in the dorsal retina of Raja. TOH and 5-HT-antisera labelled axons in the optic fiber layer of all three species but reactive ganglion cell perikarya were not identified.

Animals↗

PubMed1986

Glycogen metabolism in an amphibian retina.

The rate of incorporation of [3H]glucose into glycogen was determined in bullfrog retina incubated in vitro in dark and in the light. The rate of incorporation for glucose was found to be approximately two-fold greater in the dark than in the light, 0.108 vs. 0.061 nmol mg-1 protein min-1, respectively. The turnover rate for glycogen was found also to be approximately two-fold greater in the dark than in the light, 0.051 vs. 0.027% min-1, respectively.

Animals↗

PubMed1985

Pharmacological modification of the light-induced responses of Müller (glial) cells in the amphibian retina.

The light-evoked responses of Müller (glial) cells were monitored by intracellular recording in the isolated, superfused retina of Xenopus laevis. Müller cells had dark resting potentials of -88.5 +/- 6.9 mV and small 1-2 mV light responses of variable waveform in normal Ringer's solution. Exposure to picrotoxin (0.5-1.0 mM) greatly enhanced the light response which then consisted of depolarizing transients (Vmax 5-15 mV) at stimulus onset and offset. GABA (5-10 mM) antagonized the picrotoxin effect and suppressed the light response, whereas 2-amino 4-phosphonobutyrate (0.10-0.15 mM) blocked selectively the 'on' transient. None of these agents appreciably modified the glial cells resting potential level. On the other hand, veratrine (6-9 micrograms/ml) depolarized the Müller cell by 4-13 mV and slowed and greatly reduced the light response. These effects were antagonized by tetrodotoxin (1-4 microM) which itself reduced the light response by 30-50% without altering its shape. On the basis of these findings, we suggest that alterations in the activity of the inner retinal neurons, i.e. amacrine and ganglion cells, are primarily responsible for the drug-induced changes in the membrane potential and light-evoked responses of the Müller cell.

Aminobutyrates↗

PubMed1984

Catecholamine- and indoleamine-containing neurons in the turtle retina.

We identified a population of presumed dopaminergic amacrine cells and populations of presumed serotonergic bipolar and amacrine cells in the retina of the turtle Pseudemys scripta elegans by a combination of autoradiographic, fluorescence, and immunocytochemical techniques. Antisera directed against the dopamine-synthesizing enzyme, tyrosine hydroxylase (TOH), stained perikarya located at the border of inner nuclear (INL) and inner plexiform (IPL) layers. Processes emitted by these cells arborized in sublaminae 1, 3, and 5 of the IPL. Incubation of retinas in 10(-6) M 3H-dopamine yielded a labeling pattern identical to the staining pattern achieved with TOH antisera, but when the concentration of 3H-dopamine was increased 25-fold, both amacrine and bipolar cells are labeled. Following intraocular injection of dopamine, fluorescence micrography revealed both stained amacrine and bipolar cells. The bipolar cells had Landolt's clubs, pyriform perikarya located in the distal portion of the INL, and axons that coursed horizontally in the INL, then entered the IPL, and ramified in both its superficial and deeper layers. Although no fluorescent neuronal profiles were revealed following injection of serotonin (5HT), bipolar cells identical to those described were visualized with 5HT antisera. The intensity of bipolar cell staining with 5HT antisera was improved by preinjection of the eye with exogenous 5HT. We suggest that the bipolar cell is serotonergic, but that it also can actively accumulate dopamine. The 5HT antisera also stained a population of large amacrine cells whose processes ramified in IPL sublaminae 1, 4, and 5. The same populations of presumed serotonergic bipolar and amacrine cells were labeled following incubation of the eyecup in 10(-6) M 3H-5HT.

Animals↗

PubMed1984

The actions of gamma-aminobutyric acid, glycine and their antagonists upon horizontal cells of the Xenopus retina.

We examined the effects of gamma-aminobutyric acid (GABA) and glycine and their respective antagonists, picrotoxin and strychnine, upon the membrane potential and light-evoked responses of the type H1 horizontal cell of the Xenopus retina. This horizontal cell receives mixed input from rod and cone receptors. Under control conditions the mean membrane potential was -37.8 +/- 9.7 mV. Addition of 5 mM-GABA to the superfusate hyperpolarized the cell by 4.0 +/- 2.6 mV within 3-5 min; addition of 0.5 mM-picrotoxin depolarized the cell by 4.3 +/- 2.1 mV. Prolonged (greater than 15 min) exposures to the drugs elicited more pronounced changes in membrane potential. GABA and picrotoxin affected primarily the cone-dependent input to the H1 horizontal cell. Under dark-adapted conditions, response wave forms were essentially unaltered by the drugs, but when the horizontal cell was moderately or fully light adapted, GABA reduced and picrotoxin enhanced the cone-dependent component of its response to light. Long-term (greater than 15 min) exposures to GABA and picrotoxin elicited changes in response kinetics usually associated with dark and light adaptation, respectively. Glycine, at bath concentrations of 0.6 mM or greater, depolarized horizontal cells by 21 mV on average and reduced or abolished their light response. This action did not occur in the presence of 0.1 mM-strychnine. When all light-evoked activity was blocked by 20-40 mM-magnesium, the depolarizing action of glycine still occurred. Thus, glycine appears to act directly upon the horizontal cell membrane. Neither GABA nor glycine, nor their respective antagonists, affected the spatial extent of the horizontal cell receptive field.

Action Potentials↗

PubMed1983

Gap junctions among the perikarya, dendrites, and axon terminals of the luminosity-type horizontal cell of the turtle retina.

Gap junctions of the H1 horizontal cell of the turtle retina (Leeper, '78) were studied in thin-sectioned material and in freeze-fracture replicas. Perikaryal gap junctions were extremely restricted, 0.02-0.07 micron2 in in area, whereas those of axon terminals were much larger, most being 0.1-1.0 micron2. Both varieties, however, had the usual seven-layered appearance in thin section and measured 15 +/- 1 nm in overall width between cytoplasmic faces. Freeze-fractured views of the perikaryal junctions revealed roughly circular patches of P-face 9-nm particles and E-face pits. The axon terminal gap junctions were seen as large areas of P-face particles and E-face pits containing occasional islands of unspecialized membrane. Particle densities varied from 1,455 to 2,448 microns-2. A serial reconstruction was made of a portion of the axon terminal network in order to measure the surface areas of the axons contained therein and the fraction occupied by gap junctions. These data demonstrated that the fractional area occupied by gap junctions was roughly in inverse proportion to the area of the axon region (tuberous core vs. terminal process). It is argued that this constitutes an impedance matching device to ensure adequate current flow through the axon processes. Assuming that each P-face particle represents a connection having a conductance of 10(-10) S and given the P-face particle density and gap junctional areas determined in this report, we calculated that the gap junction distribution is adequate to account for the spatial properties of the horizontal cell axon network (Lamb, '76).

Animals↗

PubMed1983

Intracellular recording from identified photoreceptors and horizontal cells of the Xenopus retina.

Intracellular recordings were made from rods, cones and horizontal cells of the Xenopus retina. The cells under study were identified by injection of the fluorescent dye, Lucifer yellow. Rod spectral sensitivity peaked near 524 nm, that of cones near 612 nm whereas horizontal cells reflected input from both these classes of photoreceptors. No intracellular recordings were made from blue-sensitive rods (lambda max = 445 nm) nor did this rod appear to provide an input to the horizontal cell. Under dark-adapted conditions, horizontal cells had a slow waveform, a Vmax less than or equal to 18 mV and were driven by 524 nm rods only. When light-adapted, horizontal cell responses were fast, Vmax was 30-40 mV and the responses reflected only 612 nm cone input. In the mesopic state rod and cone inputs to the horizontal cell interacted non-linearly: weak green backgrounds greatly enhanced the response to a superimposed red flash compared to the red flash response on a dark field. The length constant of the horizontal cell exceeded its dendritic arbor by 2-15 fold. All of the stained horizontal cells, however, possessed a long slender axon without a terminal but which emitted periodic short branches that appeared to contact receptors.

Action Potentials↗

PubMed1983

Blue-sensitive rod input to bipolar and ganglion cells of the Xenopus retina.

Intracellular recordings were obtained from chromatic and non-chromatic bipolar cells, identified by Lucifer yellow injection in the Xenopus retina. The chromatic cells, which lacked center-surround organization, were short wavelength hyperpolarizing (lambda max 445 nm) and long wavelength depolarizing. Under photopic conditions the depolarizing component was driven by 612 nm cones, but under mesopic conditions it appeared that 524 nm rods also constituted an input to the response. The non-chromatic bipolars encountered were of the off-center (hyperpolarizing) variety, with an active antagonistic surround, and peak spectral sensitivity in the red portion of the spectrum. Extracellular recordings were obtained from color-coded ganglion cells classified as type 1 or 2 in frog retina by Maturana et al. (1966) [J. gen. Physiol. 43, 129-175] and Bäckström and Reuter (1975) [J. Physiol. 246, 79-107]. The spectral sensitivity of the long latency "on" component was matched by the density spectrum of the 445 nm rod. This response component lacked center-surround organization and showed a relatively broad area of spatial integration. In contrast, a short latency component had a spectral sensitivity matched by the 612 nm cone pigment under photopic conditions, was either "on" or "off" center, showed center-surround organization and had a relatively small area of spatial integration. We speculate that in Xenopus retina, both chromatic and non-chromatic bipolar cells provide synaptic input to the class 1,2 ganglion cell.

Action Potentials↗

PubMed1983

Rod and cone inputs to bipolar and horizontal cells of the Xenopus retina.

This report summarizes some recent studies of the Xenopus retina in which intracellular recordings were made from photoreceptors, horizontal and bipolar cells. The studied cells were identified by injection of Lucifer yellow. Rod spectral sensitivity functions conformed to the density spectrum of a 524 nm pigment, those of cones to that of a 612 nm pigment. Horizontal cell responses reflected both these classes of photoreceptor input. Rod input evoked a slow waveform, with Vmax less than or equal to 18 mV, cone input a faster waveform with Vmax = 30-40 mV. In the mesopic state the horizontal response reflected both waveforms. Rod and cone inputs to the horizontal cells appeared not to act independently, in that a steady weak green background greatly enhanced the response to a superimposed red flash, but not the reverse. A third photoreceptor type (blue-sensitive rod, Y lambda max = 445 nm) provided input to a chromatic bipolar cell which was hyperpolarized by blue light and depolarized by red light. Such chromatic bipolars had broad areas of spatial integration and lacked center-surround organization.

Adaptation, Ocular↗

PubMed1983

Retinal hexokinase: kinetic properties and the effect of cyclic 3',5'-adenosine monophosphate.

We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog (Rana catesbeiana) retinas. Seventy-three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2-deoxy-D-glucose, and mannose, but not galactose. The Km for glucose was 0.14 mM, for 2-deoxy-D-glucose, 3.6 mM. With glucose as substrate, the Vmax for particulate HK was 125-148 microM retina-1 min-1, for soluble HK, 37 microM retina-1 min-1. Product inhibition of particulate HK activity by glucose 6-phosphate was marked, whereas 2-deoxy-D-glucose 6-phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2-0.8 mM; AMP was much less effective in this regard.

Animals↗

PubMed1982

Uptake and localization of 3H-2 deoxy-D-glucose by retinal photoreceptors.

Following dark incubation of isolated retinas of Xenopus laevis in Ringer solution supplemented with 3H-2-Deoxy-D-glucose (2DG), virtually all of the uptake of the label was by the inner segments and synaptic bases of the photoreceptor cells. Autoradiographs prepared from conventionally fixed tissue showed the same cellular distribution of label as those prepared from identically incubated, unfixed, freeze-dried retinas. However, fixation removed about 77% of the total counts. This fixation-labile, soluble fraction was identified as being primarily 2DG-6 phosphate by thin-layer chromatography. The remaining insoluble fraction corresponded in distribution to glycogen grains. In cones, glycogen is stored primarily in the paraboloid, whereas in rods it is distributed throughout the inner segment and synaptic base. EM autoradiographs illustrated that these were the sites over which fixation-resistant 2DG label was localized. Measurements of radioactivity associated with extracts of retinal glycogen following 2DG incubation demonstrated that a disproportionately high fraction of total counts were associated with the glycogen fraction. We conclude that in the amphibian retina 2DG may be incorporated into glycogen.

Animals↗

PubMed1982

gamma-Aminobutyric acid antagonists decrease junctional communication between L-horizontal cells of the retina.

The antagonists of gamma-aminobutyric acid, bicuculline and picrotoxin, were found to narrow the receptive field profile of the large field horizontal cell (L1HC) in the turtle retina when added to the perfusion medium in micromolar concentrations. The coupling resistance between neighboring L1HCs was increased by bicuculline or picrotoxin. Under control conditions, the dye Lucifer yellow injected into one L1HC diffused into a large number of neighboring L1HCs; bicuculline or picrotoxin greatly restricted dye passage between these same cells. We conclude that antagonists of gamma-aminobutyric acid decrease the conductance of gap junctions between L1HCs.

Animals↗

PubMed1982

Transport and phosphorylation of 2-deoxy-D-glucose by amphibian retina. Effects of light and darkness.

We studied the uptake of 2-deoxy-D-glucose (2DG) and the synthesis of its phosphorylated product 2DG-6-phosphate (2DG-6P) by the retinas of the clawed frog (Xenopus laevis) and the bullfrog (Rana catesbeiana). Autoradiographs showed that most of the retinal 2DG uptake is by the photoreceptor layer. The 2DG accumulation by isolated Xenopus retinas was time and concentration dependent. The Kt for transport was 5.05 mM; Vmax was 6.99 X 10(-10) mol . mg-1 tissue wet weight min-1. The Km for 2DG-6P formation was estimated to be 2-3 mM and Vmax to be approximately 4 x 10(-9) mol . mg-1 min-1. 2DG uptake was inhibited competitively by glucose with a Ki of 2.29 mM. Exposure to light reduced 2DG uptake by no more than 10% as compared with dark uptake. Low sodium or ouabain (10(-4)-10(-7) M) treatment did not significantly alter 2DG uptake as compared with control retinas. In experiments upon intact, anesthetized bullfrogs, light reduced both the total amount of radioactivity acquired by the retina and the fraction of 2DG-6P present. The results are discussed in terms of the fraction of energy consumed by the retina required to maintain the photoreceptor dark current.

Animals↗