Koroku Negishi

Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas.

The localization of cells immunoreactive to a monoclonal antibody against protein kinase C (PKC) and to polyclonal antibodies against tyrosine hydroxylase (TH) was investigated in the retina of fish (carp, goldfish, dace and catfish), frog, turtle, chick and some mammalians (guinea pig, rat, cat and rabbit) by means of fluorescence microscopy. PKC-like immunoreactivity was found in dopamine (DA) or TH-like immunoreactive (IR) cells in all the species examined and also in rod bipolar cells in the fish (except for catfish), and in presumed rod bipolar cells in the other animals (except for frog and turtle). In the catfish, frog and turtle retinas, no PKC-like IR bipolar cells were found. In the rat retina, some other amacrine cells in addition to TH-like IR amacrine cells were reactive to the anti-PKC antibody. It is of interest that PKC-like immunoreactivity is commonly found in DA cells and probably in rod bipolar cells in most animal species, although the functional significance is unknown at present.

A GABA antagonist, bicuculline, exerts its uncoupling action on external horizontal cells through dopamine cells in carp retina

External horizontal cells in isolated retinas of the carp (Cyprinus carpio) were intracellularly recorded and marked with the fluorescent dye Lucifer Yellow (LY). These cells are electrically coupled via gap junctions, so that the injected dye normally diffused to neighboring cells. A GABA antagonist, bicuculline (Bcc, 20 microM), applied to the vitreous fluid beneath the isolated retina, altered the spatial properties of light-induced responses, by increasing the amplitude of responses to central spots and decreasing that of those to distant spots. Bcc also restricted to injected dye to single recorded cells. These uncoupling actions of Bcc were similar to those of amphetamine (20 microM) or dopamine (10-20 microM) and were abolished by the presence of a dopaminergic blocker, haloperidol (20-40 microM). Both the actions of Bcc and amphetamine were not observed when applied to retinas deprived of dopaminergic cells by prior destruction with 6-hydroxydopamine. Therefore, Bcc exerts its uncoupling actions on external horizontal cells indirectly through the dopaminergic system in the carp retina.

Chapter 1 The dopamine system of the teleost fish retina

In the present review, we first summarize our results of histochemical, electrophysiological and neurochemical experiments, and then relate findings reported by other investigators. Of course, our study is still in progress and recent experiments on the DA system of vertebrate retinas have been greatly advanced by others and have raised further problems to be solved

Paradoxical induction of dopaminergic cells following intravitreal injection of high doses of 6-hydroxydopamine in juvenile carp retina

Histofluorescence studies were conducted on two groups of juvenile carp (body length, 12.7 and 6.6 cm on the average, respectively) during certain periods (3-11 months) of retinal growth, to evaluate the destructive effect of 6-hydroxydopamine on dopaminergic and indoleamine-accumulating cells. Within a certain range of doses (0.1-5.0 micrograms/eye) of the neurotoxin injected intravitreally, it was found to destroy both classes of cells in a dose-dependent manner: more cells disappeared with higher doses. However, a high dose (5-25 micrograms/eye) of the neurotoxin caused paradoxical events in the retina, inducing an appearance of clustered dopaminergic cells with various soma sizes and abnormally high regional density, disturbed laminar organization, and a facilitated growth rate at the retinal margin. A preliminary examination with [3H]thymidine labelling suggests that the high dose of 6-hydroxydopamine may cause severe damage to certain classes of cells including dopaminergic and indoleamine-accumulating cells, and that a metamorphic change of precursor cells (neuroblasts) in the outer nuclear layer is responsible for the induction of such abnormal dopaminergic cells as clusters.

5‐Hydroxytryptamine Stimulates [3H]Dopamine Release from the Fish Retina

Abstract: Serotonin (5‐hydroxytryptamine, 5‐HT; 0.5 μM and above) stimulated the release of [3H]dopamine ([3H]DA) from particulate fractions of the carp (Cyprinus carpio) retina. The 5‐HT effect was dose‐ and Ca2+‐dependent, and was structurally specific. A similar response was not elicited by the other indoles (5,6‐dihydroxytryptamine, 5,7‐dihydroxytryptamine, 5‐hydroxytrypto‐phan, or 5‐hydroxyindoleacetic acid) examined. An increase in [3H]DA release was elicited by addition of 5‐HT agonists (5‐methoxytryptamine, 5‐methoxy‐N,N‐ dimethyltryptamine, and tryptamine), but not antagonized by three 5‐HT antagonists (metergolin, methysergide, and spiperone). Either DA alone or noradrenaline (0.5 mM) produced a large increase in [3H]DA release from the particulate fractions, but this action was Ca2+‐independent. Further, no significant release of [3H]γ‐aminobutyric acid could be evoked by 5‐HT (0.5 mM) under similar experimental conditions. Taken together, the present data suggest that 5‐HT stimulates [3H]DA release from the fish retina through a specific receptor‐mediated mechanism on dopaminergic terminals, but not through an exchange or nonspecific phenomenon.

So-called interplexiform cells immunoreactive to tyrosine hydroxylase or somatostatin in rat retina

The morphology of so-called interplexiform (IP) cells immunoreactive to tyrosine hydroxylase (TH) or somatostatin (SOM) in the rat retina was described in comparison with those in the carp retina. In frozen cross-sections of the rat retina, many processes of TH-like immunoreactive cells were found to extend toward the outer plexiform layer (OPL), forming a thin layer of network fibers. A few of them further extended into the photoreceptor cell layer; such fibers were never found in the carp retina. Some processes of SOM-like immunoreactive cells in the rat retina were found to travel across the inner nuclear layer and appeared to poorly develop a network at the OPL. In the carp retina, on the other hand, only one exceptional cross-section contained such an ascending process.

Dye coupling between amacrine cells in carp retina

Amacrine cells in isolated retinas of the carp (Cyprinus carpio) were intracellularly recorded and marked with a fluorescent dye, Lucifer yellow. On occasion, dye coupling was found to occur between amacrine cells when the dye was iontophoretically injected into an amacrine cell, generating one of the transient or sustained types of photoresponse. Dye-coupled cells in the vicinity of the marked cell were very similar to the marked cell in soma shape and dendritic stratification. Dye diffusion is assumed to take place at gap junctions between dendrities of amacrine cells which belong to a population of similar type cells in morphology and possibly in function.

An electrophysiological study on the cholinergic system in the carp retina.

The effects on spike discharges of acetylcholine chloride (ACh), applied electrophoretically at the inner plexiform layer, were examined in air-exposed and superfused preparations of the isolated carp retina. Most of spike-generating units examined (147 of 204 units; 72%) were sensitive to electrophoretically applied ACh. Among the ACh-sensitive ones, 125 units (85%), including all ON-center, most ON-OFF and two-thirds of OFF-center units, were activated, whereas 22 units (15%), including mainly OFF-center units, were suppressed by the agent. In retinas superfused with a medium containing 20 mM M2+ and 0.5 mM Ca2+, units ceased in their spontaneous and light-induced discharges, but they were activated by electrophoretically applied ACh. The ACh-induced activation was reduced in magnitude or abolished during an exposure of the retina to a medium containing hexamethonium chloride (25 micrometer), whereas the ACh-induced suppression was reduced by atropine sulfate (25 micrometer) more effectively than by hexamethonium. Therefore, the results suggest that nicotinic receptors are involved in the ACh-activation, whereas both, but predominantly muscarinic, receptors may participate in the ACh-suppression of spike discharges in the carp retina.

Two types of lamprey retina photoreceptors immunoreactive to rod- or cone-specific antibodies.

The localization of structures immunoreactive to antisera against opsin (OPS; a specific marker for rods) and against visinin (VIS; a specific marker for cones in the vertebrate retina) was investigated in the retina of the river lamprey (Lampetra japonica) by means of fluorescence microscopy. Different immunoreactive structures were found with the two antisera. A vitreadly located structure in the outer nuclear layer displayed OPS-like immunoreactivity, while a scleradly located one exhibited VIS-like reactivity. Thus, the two antisera applied to the retina of the river lamprey permit the distinguishing of rod and cone immunoreactive photoreceptors such as occurs typically in all vertebrate classes. The former appears in the outer segments of a type of rod cells, and the latter in the cell bodies and axons of cones.

GABAergic Inhibition on Dopamine Cells of the Fish Retina: A [3H]Dopamine Release Study with Isolated Cell Fractions

Abstract: Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When γ‐aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 μM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.