Berndt Ehinger

The interplexiform cell system - I. Synapses of the dopaminergic neurons of the goldfish retina

Interplexiform cells are a class of retinal neuron that extends processes widely in both plexiform layers. In goldfish they contain dopamine and readily take up certain biogenic amines. Two of these amines, 6-hydroxyopamine (6-HDA) and 5, 6-dihydroxytryptamine (5,6-DHT), induce fine structural changes in the neurons that accumulate them, allowing the processes of the cells to be recognized by electron microscopy. Typically, the synaptic vesicles within the processes show electron-dense cores. The terminal cytoplasm may also show increased density, as may the cellular and cytoplasmic membranes, presumably an indication of degenerative changes induced by the drugs. 5, 6-DHT gives more readily observable changes than 6-HDA but labels both dopaminergic and indoleamine-accumulating neurons. The terminals of the indoleamine-accumulating terminals were therefore removed by intraocular injections of 5, 7-dihydroxytryptamine (5, 7-DHT) prior to the labelling with 5, 6-DHT. This procedure allowed an analysis of the dopaminergic terminals without interference by the terminals of the indoleamine-accumulating cells. The dopaminergic neurons were found to make synapses of the conventional type. In the outer plexiform layer they contacted both external horizontal cells and bipolar cell dendrites, but not hotoreceptor terminals or intermediate (rod) horizontal cells. No synapses onto the dopaminergic processes were found in the outer plexiform layer despite an extensive search. In the inner plexiform layer the dopaminergic processes were observed to be both pre- and postsynaptic to amacrine cells and their processes. No synaptic contacts between dopaminergic processes and bipolar cell terminals or ganglion cell dendrites were seen. We conclude that the dopaminergic interplexiform cells provide a centrifugal pathway for information flow in the retina from inner to outer plexiform layer.

Autoradiography of some suspected neurotransmitter substances: GABA glycine, glutamic acid, histamine, dopamine, and pl-dopa

The uptake of GABA, glycine, glutamic acid, aspartic, l-DOPA, dopamine and histamine into the rabbit retina in vivo and in vitro was studied autoradiographically. GABA and glycine accumulated in cells that had in the main the position and spread of amacrine cells and also in some ganglion cells. Radioactivity appeared in Mu¨ller cells and retinal pigment cells after exposure of the retina to aspartic or glutamic acid in vivo. After exposure to histamine in vivo, radioactivity appeared in the retinal pigment cells and diffusely in the retina. l-DOPA and dopamine were taken up into cells with the position of the adrenergic retinal neurons. The experiments demonstrate that in central nervous tissue such as the retina, certain classes of cells preferentially accumulate glycine or GABA, presumably into two different sets of cells. For several reasons, it is less probable that ganglion cells operate with either glycine or GABA as neurotransmitter; the results thus suggest that the preferential uptake of either substance into a cell is not necessarily a sign of its being a neurotransmitter in the particular cell. However, reasons are given for presuming GABA to be the neurotransmitter of certain amacrine cells, and the results also suggest that a similar role for glycine is worthy of further consideration.

A method for differentiating dopamine from noradrenaline in tissue sections by microspectrofluorometry.

Fluorophores induced from noradrenaline and dopamine in tissue sections by treatment with formaldehyde can be separated by their different behavior upon HCl treatment. The noradrenaline fluorophore converts to a fully aromatic isoquinoline while the dopamine fluorophore remains in the form of a nonquinoidal dihydroisoquinoline. The two fluorophores have readly distinguishable excitation spectra. The procedure has been tested both in model systems and in tissue sections. The microspectrofluorometer used is described.

Indoleamine-accumulating neurons in the retina of rabbit, cat and goldfish

SummarySpecial neurons accumulating indoleamines have been detected in the retina of rabbit, cat and goldfish. They have their perikarya in the inner-most cell row of the inner nuclear layer, among the amacrine cells, and send their processes to various parts of the inner plexiform layer. The distribution of the processes is different in the different animals investigated. The neurons do not correspond to the previously known dopaminergic retinal neurons, which have a different distribution of their terminals and which can be demonstrated with a specially developed technique, simultaneously with the indoleamine-accumulating neurons.

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

SummaryThe relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.

Glial and neuronal uptake of GABA, glutamic acid, glutamine and glutathione in the rabbit retina.

Abstract Rabbit retinas were exposed to radioactive glutamic acid, glutamine, GABA or glutathione either by injecting the substances intravitreally in vivo or by incubating the retina in them in vitro. There is both a glial and a neuronal uptake of GABA both in vivo and in vitro. The radioactivity is well retained in neurons, but more rapidly lost from the glia, apparently at least partly by metabolism. Therefore, the glial distribution of radioactivity is seen at short times after intravitreal injections or immediately after short (15 min) incubations, whereas the neuronal type of distribution is seen at longer times after the injection or with longer incubation times, and particularly so when the tracer is excluded during the prolongation. Glutamic acid is in vivo mainly taken up by glia. In vitro there is also uptake of radio-activity into horizontal cells but it is not known whether this represents glutamic acid or a metabolite. Only after prolonged incubations is any uptake seen in amacrines, which in rabbits are the neurons which most likely operate with GABA as transmitter. Neither glutamine nor glutathione give any accumulation of radioactivity in amacrines (except after prolonged incubations). Moreover, [1-14C]GABA will label the same number of amacrines as [U-14C]GABA. The experiments thus fail to support the hypothesis that the GABA skeleton is rapidly transported between glia and neurons in the form of glutamine or glutamic acid.

Adrenergic neurons in teleost retina.

SummaryThe adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.

Cellular location of the uptake of some amino acids into the rabbit retina

The uptake of 20 different amino acids into the rabbit retina was studied autoradiographically in vivo after intravitreal injections or in vitro after incubations in order to obtain results pertinent to the autoradiographic procedure for demonstrating neurones putatively operating with certain amino acids as neurotransmitters. The distribution of radioactivity in the retina is similar after the application of most amino acids. Radioactivity accumulates in the retinal ganglion cells and to a lesser extent (in falling order) in glial cells, pigment epithelium, nerve cells of the inner nuclear layer, and other nerve cells. The amino acids are rapidly metabolized. There are only minor autoradiographic variations with experiments in vivo or in vitro. Five of the amino acids studied display divergent behavior. In addition to the uptake into the retinal ganglion cells (regarded as a nonspecific uptake) certain cells with the position of amacrines accumulate glycine, GABA and β-alanine. These amino acids seem to be taken up into protecting stores. Glycine and GABA are regarded as putative inhibitory retinal neurotransmitters. The possibility that β-alanine is also a neurotransmitter is discussed. It is tentatively concluded that at present it cannot be regarded as a putative neurotransmitter, but that it probably only mimics the behavior of an unknown neurotransmitter. Glutamic and aspartic acids also differ autoradiographically from the majority of the amino acids. They are almost exclusively taken up by glial cells and only to a minor extent by retinal neurones of any type. They are relatively rapidly metabolized. If they are neurotransmitters, it seems possible that they are inactivated by glial uptake.

Uptake of certain possible neurotransmitters into retinal neurons of some mammals

The uptake of glycine, GABA, and β-alanine was studied in rat, guinea-pig, cat, monkey and human retinas. The human retinas were studied in vitro only, the others both in vivo and in vitro. Either in vivo or in vitro [3H]glycine was aceumulated by certain cells which presumably are a type of amacrine cells. Glycine was not significantly metabolized under the conditions of the experiments. There was no significant variation between the species with this amino acid. [3H]GABA was preferentially accumulated by a type of amacrines in vivo. In rats and monkeys there was also a variable uptake into Muller cells already in vivo, and this uptake often disguised the neuronal uptake into amacrines. [3H]GABA in vitro gave predominantly glial uptake in all species except in cats and guinea-pigs where radioactive amacrines were still seen. [3H]β-alanine gave results similar to [3H]GABA. [3H]Glutamic acid produced predominantly glial uptake either in vivo or in vitro. Rods but not cones became somewhat radioactive in vitro. The results emphasize that particularly the GABA uptake varies significantly between different species.

Adrenergic and cholinesterase-containing neurons of the heart

SummaryThe adrenergic and acetylcholinesterase-containing nerves of the hearts of mice, rats, guinea-pigs, rabbits, and cats were studied. The fluorescence technique of Falck and Hillarp was used for the demonstration of adrenergic nerves, whereas a modified Koelle cholinesterase technique was used for the cholinesterase-containing nerves. The inhibitors used were Mipafox, iso-OMPA and Nu 683. Microspectrofluorometry was used to identify the structures containing dopamine.Adrenergic as well as acetylcholinesterase-containing fibres were found in all parts of the heart, most abundantly in the atria. Dense nerve plexa supplied the sinoarial and atrioventricular nodes. There was a plexus of both fibre types in the endocardium and on the atrial side of the valves. In the valves, it could be shown that adrenergic and cholinesterase-containing fibres ran closely parallel to each other. Indirect evidence suggested that this applies also to the myocardium.No nerve fibres containing dopamine were revealed in the microspectrofluorometer. The dopamine previously found in the atria seems, instead, to be situated in so-called small intensely fluorescent cells.No adrenergic ganglion cells were found in the heart despite extensive search. The vagus of rabbits was found to contain only few adrenergic preterminals.