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Biomedical subjects

R Weiler

Publications and source records attributed to R Weiler.

At least 19 recordsLinked to original sources
PubMed1996

Quantitative anatomy, synaptic connectivity and physiology of amacrine cells with glucagon-like immunoreactivity in the turtle retina.

Although a wide variety of neuropeptides have been localized in vertebrate retinas, many questions remain about the function of these peptides and the amacrine cells that contain them. This is because many of these peptidergic amacrine cells have been studied using only immunocylochemical techniques. To address this limitation, the present study used a combination of quantitative anatomy, biochemistry and electrophysiology to examine amacrine cells in the turtle retina that contain the neuropeptide glucagon. In the turtle retina, there is a small population of 2500 glucagonergic amacrine cells, which probably represents < 1% of the total number of amacrine cells. Circular distribution statistics indicated that many of these tristratified amacrine cells had asymmetric dendritic arborizations that were radially oriented toward the retinal periphery. The cells were found to have similar dendritic coverage factors, to be distributed in a non-random arrangement in all regions of the retina, and to peak in density in the visual streak region. Electron microscopic studies indicated that glucagonergic amacrine cells made synaptic contacts primarily with other amacrine cells, and small numbers of bipolar cells. The synaptic inputs and outputs were balanced in the inner strata of the inner plexiform layer, and were biased toward synaptic outputs in the outer strata of the inner plexiform layer. These contacts involved small unlabelled synaptic vesicles, and not the large labelled dense core vesicles also found in these neurons. The biochemical studies indicated that glucagon could be released from the retina in a calcium dependent manner by high potassium stimulation. The electrophysiology found no color opponency, and the glucagonergic amacrine cells gave sustained hyperpolarizing responses to small stimulation spots and had antagonistic surrounds. The results of these studies suggest that there are significant regional specializations of glucagonergic amacrine cells, and that they may provide OFF-modulation in interactions between the ON-and OFF-centre visual pathways in the turtle retina.

Animals↗
PubMed1995

Retraction of spinule-type neurites from carp retinal horizontal cell dendrites during dark adaptation involves the activation of Ca2+/calmodulin-dependent protein kinase II.

The formation of spinules at the terminal dendrites of retinal horizontal cells with the onset of light and their subsequent retraction during darkness is a remarkable example of synaptic plasticity where sensory experience modifies reversibly, and on a time scale of minutes the ultrastructure of synaptic connectivity. The signals and the subsequent intracellular cascades underlying the prominent morphological alterations are only partially understood. We show here that lowering the external calcium concentration did prevent dark- and AMPA-induced retraction of spinules in a eyecup preparation. Furthermore, spinule retraction was prevented in vivo by the injection of calmidazolium, an inhibitor of calmodulin, into the eyeball, and also by the injection of KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase (CaMkII). We conclude that local Ca2+ influx through AMPA-gated channels followed by activation of CaMkII is an important step for spinule retraction during dark adaptation. The phosphorylation patterns of phosphoproteins derived from purified horizontal cells was affected by the inhibitors of calmodulin and CaMkII respectively. Some of the affected phosphoproteins appeared to be cytoskeleton-associated proteins, including GAP-43. Based on these observations, a putative scenario for the retraction of spinules is proposed.

Animals↗
PubMed1995

Phorbol ester binding sites in the fish retina: correlation with stimulation of endogenous phosphorylation and protein kinase C activation.

The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritated phorbol 12,13-dibutyrate ([3H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [3H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 nM and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [3H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of 32P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.

Animals↗
PubMed1995

Ethambutol alters spinule-type synaptic connections and induces morphologic alterations in the cone pedicles of the fish retina.

PURPOSE: Ethambutol can cause optic neuropathy and deficiencies in color-opponent visual processing in patients treated for tuberculosis. In fish, Ethambutol induces color vision deficiencies similar to those observed in humans and affects color coding in retinal ganglion cells. Color opponency in fish is mainly mediated by a horizontal cell feedback onto cones thought to be provided by spinules. The authors examined whether Ethambutol affects spinules and is, therefore, able to alter color processing at a distal stage, that is, at the first synaptic connection within the retina. METHODS: Ethambutol was injected into the vitreous of either dark- or light-adapted fish. After drug application, fish were held under different illumination conditions. Thereafter, the retinas were dissected and prepared for electron microscopy. Ultrathin tangential sections of retinas were examined at the level of the outer plexiform layer. RESULTS: In already light-adapted retinas, a high dose of Ethambutol (10 mM) reduced the number of spinules by 30%. Ethambutol application in the dark with subsequent light adaptation resulted in severe dose-related inhibition of light-induced spinule formation. In these experiments, low doses (0.1 mM) of Ethambutol caused 40% inhibition, and high doses (10 mM) caused 70% inhibition. Besides affecting spinules, Ethambutol occasionally induced a degeneration of cone pedicles. This neurotoxicity only occurred in cones exposed to light. CONCLUSIONS: Results show that Ethambutol alters synaptic connections between horizontal cells and cones in a dose-related fashion; Ethambutol treatment can be toxic for cone pedicles and can cause their degeneration; and the rod pathway is not affected by the drug. This indicates that Ethambutol influences the color-coding process already at the level of the cone-horizontal cell synapse.

Animals↗
PubMed1994

Involvement of metabotropic and ionotropic glutamate receptors in inositol polyphosphate formation in carp retinal slices.

The contribution of ionotropic and metabotropic glutamate receptors to inositol polyphosphate accumulation in carp retinal slices was investigated using myo-[2-3H]inositol prelabelling. In the presence of the glutamate agonists quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and trans-(+/-)-1-amino-1,3-cyclopentane-dicarboxylic acid (t-ACPD), formation of [3H]inositol phosphate was significantly increased in a dose-dependent manner, with EC50 values of 350 nM, 1.5 microM and 10 microM respectively. The complete AMPA-induced response and a large component of the quisqualate-induced response were inhibited in a competitive manner when the ionotropic antagonist 6-cyano-7-nitroquinoxalin- 2,3-dione (CNQX) was present. Furthermore, the remaining level of quisqualate-induced [3H]inositol phosphate formation closely matched that produced by ACPD alone, and coincubation of AMPA and ACPD showed additive effects, suggesting that the quisqualate-induced response resulted from coactivation of metabotropic and ionotropic glutamate receptors. The ionotropic component was partially reduced in the presence of cobalt, suggesting indirect effects resulting from synaptic interactions. We could exclude indirect effects through depolarization-induced release of other neurotransmitters. Only serotonin (EC50 1 microM) and carbachol (at a concentration of 1 mM) stimulated [3H]inositol phosphate formation, but their antagonists did not affect the quisqualate response and coactivation with quisqualate and serotonin or carbachol resulted in additive effects. The ionotropic component was completely suppressed when Ca2+ was omitted from the medium and cobalt was present. This makes it likely that the ionotropic component resulted from Ca2+ entry through AMPA-gated channels and subsequent Ca(2+)-dependent activation of phospholipase C.

Animals↗
PubMed1994

Threshold and chromatic sensitivity changes in fish cone horizontal cells following prolonged darkness.

The light-evoked responses of L-type cone horizontal cells in the teleost retina were studied following a prolonged period of complete darkness. Intact, isolated white perch retinas were superfused in complete darkness for more than 90 min, following which horizontal cells were impaled without the aid of any light flashes. Following this prolonged darkness, L-type cone horizontal cell light responses to dim and bright full-field stimuli were slow and small in amplitude and response duration to bright stimuli was considerably longer than stimulus duration. In addition, absolute threshold was 2 log units lower than typical for cone horizontal cells and spectral sensitivity to shorter wavelengths was increased. Following bright light stimulation, light responses became more transient and increased in amplitude, reaching 40-50 mV to bright flashes. Moreover, absolute threshold increased and responses to spectral stimuli were similar to those observed typically for L-type cone horizontal cells after light-sensitization. These results suggest that following prolonged darkness, cone input to cone horizontal cells is reduced and rod input is present.

Animals↗
PubMed1994

pH-gated dopaminergic modulation of horizontal cell gap junctions in mammalian retina.

Horizontal cells mediate lateral inhibition in the outer retina, and this process is dependent on electrical coupling through gap junctions, giving rise to receptive fields that are much wider than the dendritic fields. This study on rabbit retina shows that the permeability of the gap junctions between A-type horizontal cells, as assessed by Lucifer yellow dye coupling, is modulated by dopamine through a D1 receptor linked to adenylate cyclase. Both exogenously applied dopamine and endogenously released dopamine uncoupled the horizontal cells, but the effect was pH-gated whereby it occurred only at an extracellular pH 7.2 +/- 0.05. The horizontal cells also uncoupled in acidic media (pH 7.0 or below) in the absence of dopamine. Our results show that horizontal cell coupling in the mammalian retina is regulated by both dopamine and pH. Given that the pH in the outer retina varies with the metabolic activity of the photoreceptors, these results suggest that ambient light conditions could gate the activity of neurotransmitters through pH-sensitive mechanisms.

Adenylyl Cyclases↗
PubMed1993

The marker for nitric oxide synthase, NADPH-diaphorase, co-localizes with GABA in horizontal cells and cells of the inner retina in the carp retina.

NADPH-diaphorase histochemistry selectively stained discrete populations of neurons contributing to the inner plexiform layer of the carp retina. In addition H1 horizontal cells contributing to the outer plexiform layer were labeled. In these cells as in a subpopulation of the labeled cells in the inner retina, NADPH-activity co-localized with GABA immunoreactivity. NADPH-activity is a direct marker for NO synthesis and it is concluded that NO might be an important mediator of light-dependent adaptational processes in the outer retina.

Amino Acid Oxidoreductases↗
PubMed1993

Ionotropic non-N-methyl-D-aspartate agonists induce retraction of dendritic spinules from retinal horizontal cells.

Horizontal cells invaginate the photoreceptors in the retina and form reciprocal synaptic connections in the cone pedicles. In fish retina the pattern of synaptic connections is plastic and modulated by the ambient light conditions. Numerous dendritic spinules protrude from the terminal horizontal-cell dendrites into the cone pedicle when the retina is light-adapted and are retracted during dark adaptation. The retraction of spinules can be induced during maintained illumination by an injection of the putative cone transmitter L-glutamate or its analogue kainic acid into the vitreous humor. The formation and the retraction of spinules have a time course of minutes. Activation of protein kinase C through phorbol esters initiates the formation of spinules, but the retraction has not yet been linked to a specific second messenger. Herein we report that physiological concentrations of the glutamate analogs quisqualic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induce retraction of spinules during maintained illumination. (+/-)-trans-1-Amino-1,3-cyclopentanedicarboxylic acid, an agonist for the metabotropic quisqualic acid receptor, was without effect on spinule retraction. N-Methyl-D-aspartate and L-2-amino-4-phosphonobutyric acid, agonists at other types of glutamate receptors, were also without any effect. The effects of the active agonists persisted when synaptic transmission was blocked. In the presence of the ionotropic quisqualate receptor antagonist 6-cyclo-7-nitro-quinoxaline-2,3-dione the effects of all active agonists were blocked. These results demonstrate that activation of ionotropic quisqualate receptors on the horizontal-cell membrane can induce dendritic spinule retraction, a process associated with dark adaptation.

Aminobutyrates↗
PubMed1993

In vitro phosphorylation in isolated horizontal cells of the fish retina: effects of the state of light adaptation.

Horizontal cells, which are second-order neurons of the vertebrate retina, exhibit synaptic plasticity governed by light and dark adaptation. We have investigated the alterations in the protein phosphorylation patterns of isolated carp (Cyprinus carpio) horizontal cells in relation to their state of light adaptation by using an in vitro phosphorylation assay and compared the resulting data with protein synthesis patterns of the whole retina. Phosphoproteins and [35S]methionine-labelled proteins were analysed by one- and two-dimensional gel electrophoresis followed by autoradiography. The state of light adaptation significantly affected the in vitro phosphorylation of horizontal cell proteins with molecular weights of 68, 56/58, 47, 28 and 15 kDa, but had no effect on the protein synthesis of retinal proteins. In the light the most prominent increase of 32P incorporation was observed in the 47 kDa protein. The biochemical properties of this protein closely resembled those of the growth-associated GAP-48, found in the fish retina. In addition, the phosphorylation of horizontal cell homogenates in the presence of protein kinase activators such as cyclic AMP, calcium, calmodulin and phospholipids revealed that horizontal cells of the fish retina contain cyclic AMP-, calcium/calmodulin- and calcium/phospholipid-dependent protein kinase activity resulting in the phosphorylation of several horizontal cell proteins, including the phosphoproteins which were affected by the state of light adaptation.

Adaptation, Physiological↗
PubMed1993

Spinule-type neurite outgrowth from horizontal cells during light adaptation in the carp retina: an actin-dependent process.

Dendrites of horizontal cells in the carp retina which invaginate the cone pedicles form numerous spinules during light adaptation. We have analyzed the contribution of cytoskeletal elements to this process. Isolated horizontal cells and frozen sections were screened with phalloidin for the existence of F-actin. F-actin was present in all types of horizontal cells and particularly enriched in the distal parts of the dendrites. Electron microscopical analysis demonstrated that interruption of the F-actin polymerization with cytochalasin B inhibited the formation of spinules during light adaptation. The persistence of spinules was also affected. Cytochalasin B also prevented the light-independent, phorbol ester-induced formation of spinules. Cytochalasin B only affected the morphology of the lateral, spinule-forming dendrites of cone horizontal cells within the cone pedicles, leaving the central, non spinule-forming dendrites of cone horizontal cells and the processes of rod horizontal cells within rod spherules unaffected. Whereas cytochalasin B prevented the protrusion of spinules, the spinule-associated membrane densities were only slightly affected. The two main characteristics of spinules, protrusion and membrane densities are therefore independently regulated processes.

Actins↗
PubMed1992

Effects of background illuminations on the receptive field size of horizontal cells in the turtle retina are mediated by dopamine.

Intracellular recordings from luminosity-type horizontal cells of the turtle retina were used to analyze the effects of steady and flickering background illumination on the size of their receptive fields. Both types of background illumination reduce the size of the receptive field to about the same extent. The reduction seems largely due an increase in the coupling resistance between horizontal cells. The effects of both types of background illumination are sensitive to the dopamine antagonist fluphenazine. This suggests that steady and flickering illuminations stimulate the release of endogenous dopamine.

Animals↗
PubMed1992

Synaptic pathology in Alzheimer's disease: immunological data for markers of synaptic and large dense-core vesicles.

We have analysed several markers for small synaptic vesicles (synaptin-synaptophysin, p65 and SV2) and large dense-core vesicles (chromogranin A, secretogranin II/chromogranin C) in the brains of patients with Alzheimer's disease, and normal controls by immunoblotting and immunohistochemistry. In comparison to age-matched controls the levels of all three synaptic vesicle markers were decreased in temporal cortex of Alzheimer patients. On the other hand, the levels of chromogranin A were increased, and those of secretogranin II lowered. This resulted in a significant increase of the ratios of chromogranin A to synaptophysin, p65 or SV2 and of that for chromogranin A to secretogranin II. These increases were significantly correlated to clinical severity of dementia and extent of neuropathological changes. By immunohistochemistry a high percentage of senile plaques was found to contain chromogranin A-reactive dystrophic neurites, whereas synaptophysin reactivity within plaques was rare. These results indicate that the number of synaptic vesicles is lowered in Alzheimer's disease, and that one component of large dense-core vesicles, i.e. chromogranin A, is elevated. We, thus, suggest that in Alzheimer's brain distinct changes occur for both types of synaptic organelles.

Aged↗
PubMed1992

Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina.

In mammalian retina, the rod bipolar cells synapse on the AII amacrine cells, which are therefore the third-order neurons in the rod-signal pathway. The AII amacrine cells are connected by gap junctions, both to each other and to fourth-order, On-center cone bipolar cells. They also receive synaptic input from the dopaminergic amacrine cells, and in this study, we investigated whether dopamine modulates the permeability of the gap junctions between AII amacrine cells in the isolated rabbit retina. The small biotinylated tracer Neurobiotin was injected into nuclear yellow-labeled AII cells under direct microscopic control. The extent of tracer coupling to neighboring AII cells, 40-60 min after Neurobiotin injection (0.5 nA for 60 sec), provided a standard measure of the permeability of the homologous gap junctions. Under control conditions, individual AII amacrine cells were coupled to 73 +/- 15 neighboring cells, and this was unaffected by changes in pH from 6.6 to 7.8. Exogenous dopamine significantly reduced the tracer coupling at concentrations as low as 10 nM (26 +/- 16 cells), with the effect increasing with dopamine concentration up to 10 microM (6 +/- 4 cells). The uncoupling effect of dopamine was both blocked by the selective D1 antagonist SCH-23390 (10 microM) and mimicked by the specific D1 agonist SKF-38393 (500 microM). Moreover, the AII amacrine cells were also uncoupled when the retina was incubated in forskolin (60 microM) and isobutylmethylxanthine (200 microM). Taken together, these results indicated that the uncoupling was mediated by a D1-like receptor that stimulates cAMP production. Although the selective D1 antagonist on its own did not increase tracer coupling, suggesting that there was little release of endogenous dopamine in the superfused photo-bleached retina, veratridine-evoked release of endogenous transmitters did uncouple the AII amacrine cells, and this effect was blocked by the specific D1 antagonist.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Synaptin/synaptophysin, p65 and SV2: their presence in adrenal chromaffin granules and sympathetic large dense core vesicles.

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.

Adrenal Medulla↗
PubMed1991

Protein kinase C mediates transient spinule-type neurite outgrowth in the retina during light adaptation.

Light and dark adaptation of the teleost retina is accompanied by a remarkable morphological rearrangement of the synaptic connections between photoreceptors and second-order neurons: during light adaptation, numerous new neurites, the so-called spinules, arise from the terminal dendrites of horizontal cells invaginating the cone pedicle, and during dark adaptation, these spinules are retracted. The formation of these spinules is paralleled by the appearance of color opponency in horizontal and ganglion cells, which led to the suggestion that these spinules are the site of the inhibitory synapses in the negative feedback loop between cones and horizontal cells. The formation of the spinules in the light and their disappearance in darkness have a time course of minutes and are modulated by the neurotransmitters dopamine and glutamate, respectively. Neurotransmitters can modulate neuronal processing through a variety of second messengers that activate protein kinases, resulting most commonly in protein phosphorylation. Herein we report that activation of protein kinase C by phorbol esters promotes the formation of new horizontal-cell spinules in animals kept in the dark. Partial inhibition of protein kinase C activation with sphingosines prevents the formation of new spinules during light adaptation but does not affect established spinules. The spinule-forming effect of phorbol esters is not mediated by dopaminergic neurons, since the effect is also seen in retinas depleted of dopaminergic neurons. Phorbol esters also initiate the formation of spinules in synaptically isolated horizontal cells, demonstrating that they have a direct action on these cells. In addition, isolated horizontal cells have substrate proteins that are phosphorylated in a protein kinase C-dependent manner.

Adaptation, Physiological↗
PubMed1991

Short-term potentiation of off-responses in turtle horizontal cells.

Depolarizing responses to light off were studied in turtle horizontal cells using intracellular recording in the everted eyecup preparation. In many cells the off-response showed two components (fast and slow) which could overshoot beyond the steady-state dark level. The peak amplitudes of the fast and slow components increased with increasing duration of the light stimulus. A similar enhancement of the off-responses could also be produced by repetitive stimulation with brief flashes. However, the degree of enhancement produced by repetitive stimulation was greater than could be produced by increasing stimulus duration, and the latency of the onset of depolarization was longer, suggesting that the enhancement produced by repetitive stimulation involves an additional mechanism. Dramatic enhancement of the off-response by stimuli which did not affect the on-response during light indicates that the off-response may contain information not present in the on-response. The fast component of the off-response was suppressed to a greater degree than other components by reducing extracellular calcium or in the presence of 500 microM cobalt, suggesting that this component may involve a calcium current.

Animals↗