Multiple neurotransmitter systems influence the release of adenosine derivatives from the rabbit retina

Rabbit retinac preloaded with [³H]adenosine were superfused in vitro and the effect of neurotransmitter agonists and antagonists on the release of [³H]purines was studied. Glutamic acid, aspartic acid, kainic acid (KA), quisqualic acid (QUIS) and acid (NMDA) all stimulated the efflux of [³H] labelled and endogenous purines. Their effect was reduced in a Ca²⁺-free medium except when using a high concentration (100 μM) of KA, QUIS and NMDA. The effect of aspartic acid and of NMDA were blocked by 2-amino-7-phosphono-heptanoic acid (APH) and 2-amino-5-phosphono-valeric acid (APV). Carbachol also increased the release of adenosine-derived radioactivity and this effect was reduced by the removal of Ca²⁺ and by pretreatment with atropine. τ-Aminobutyric acid (GABA) and muscimol, induced a small increase in the release which was Ca²⁺-dependent and was blocked by bicuculline and picrotoxin. Dopamine elicited an increase in the release which was partially reduced in a Ca²⁺-free medium and was blocked by haloperidol. Glycine and 5-hydroxytryptamine (5-HT) also induced small but significant increases. The neurotransmitter antagonists had an effect of their own. Superfusion with APH and APV depressed the outflow of radioactivity whereas bicuculline, picrotoxin, strychnine and haloperidol enhanced it. The K⁺-evoked release of [³H]purines was reduced by haloperidol and by 5-HT. The observations indicate that stimulation of several important neurotransmitter receptors in the retina elicits the release of adenosine derivatives. The results with the antagonists also suggest that purines are continuously released as a result of a tonic activation of the respective membrane receptors.     

Modulation of [3H]dopamine release from rabbit retina

The calcium-dependent release of [3H]dopamine ([3H]DA) elicited by field stimulation or potassium is modulated through activation of stereoselective inhibitory DA autoreceptors of the D-2 subtype that are pharmacologically different from the D-1 DA receptor subtype linked to the stimulation of adenylate cyclase (EC 4.6.1.1). The D-2 DA autoreceptors appear to be endogenously activated by DA because DA receptor antagonists such as S-sulpiride increased the stimulation-evoked release of [3H]DA. Nanomolar concentrations of norepinephrine (NE) and epinephrine (E) inhibited in a concentration-dependent manner the electrical stimulation-evoked release of [3H]DA. The inhibitory effect of these catecholamines was not modified by S-sulpiride, which, on the contrary, selectively antagonized the inhibition of [3H]DA release elicited by exogenous DA. Phentolamine or (+/-)-propranolol did not affect the release of [3H]DA from rabbit retina. The alpha antagonist phentolamine competitively antagonized the inhibitory effect of both NE and E, which suggests that these catecholamines activate alpha receptors in retina. The decrease by catecholamines of the calcium-dependent release of [3H]DA appears not to involve beta adrenoceptors because their inhibitory effect was not modified by propranolol. Under identical experimental conditions (i.e., nomifensine, 30 microM), serotonin did not modify the stimulated release of [3H]DA. In conclusion, in the rabbit retina, DA autoreceptors of the D-2 subtype appear to modulate endogenously released DA whereas inhibitory presynaptic alpha receptors might be of pharmacological importance as sites of action for retinal or blood-borne catecholamines.     

Calcium dependent release of acetylcholine and gamma-aminobutyric acid from the rabbit retina.

The concurrent release of endogenous ACh and GABA from the retina (in the presence of physostigmine) was measured using either an eye-cup preparation in rabbits anaesthetized with urethane or isolated rabbit retinas. There was a spontaneous resting release of ACh and GABA from the dark adapted retina of ca 5 and 160 pmol min-1 respectively. Stimulation of the initially dark adapted retina in vivo with flickering light (0.1-20 Hz) increased the release of ACh by up to 5 times the spontaneous resting release but did not cause a detectable increase in GABA release. The maximum light-evoked release of ACh was about 24 pmol min-1/retina and occurred at a frequency of 10 Hz. However, the maximum release of ACh per flash occurred at 0.1 Hz at which frequency the average ACh release per flash from one amacrine cell was ca 2.35 x 10(-18) mol. Exposure of the retina to the potent inhibitors of GABA uptake, SKF89976A and SKF100330A markedly reduced the resting release of ACh and abolished the light-evoked release of ACh but did not enable a light-evoked release of GABA to be detected. Bicuculline blocked the inhibitory actions of both SKF89976A and SKF100330A on ACh release but the combination of bicuculline and uptake inhibitor did not result in a light-evoked release of GABA. In contrast, KCl (20 mM) applied locally to the retina in vivo resulted in the release of both ACh and GABA (61 and 2.6-fold respectively). KCl (20 mM) also evoked large increases in ACh and GABA release from isolated rabbit retinas in room light (13.5 and 3.4-fold respectively). The K-evoked release of ACh and GABA from the rabbit retina both in vivo and in vitro was calcium dependent. These experiments are the first in which endogenous ACh and GABA release from the retina have been simultaneously measured and suggest that the release mechanisms for these transmitters are fundamentally similar.     

Aldehyde oxidase from rabbit liver: specificity toward purines and their analogs

Aldehyde oxidase (EC 1.2.3.1) plays a role in the oxidation of aromatic heterocyclic compounds ingested by some higher vertebrates. To better understand this function, the specificity of the rabbit liver enzyme toward purines and their analogs was quantitatively studied. The chemical nature of the 6-substituent of purine markedly influenced substrate efficiency (Vmax/Km). Substituents that were hydrophobic were generally favorable. There was a correlation between the degree of hydrophobicity and the tightness of binding. 6-Substituents that were strongly electron-withdrawing also enhanced substrate efficiency. 6-Hydroxy and 6-amino substituents virtually obliterated substrate activity. In contrast, 2-hydroxypurine and 2-aminopurine were efficiently oxidized. 2,6-Disubstitution of purine was much less favorable than either 2- or 6-monosubstitution. N-Substitution of purines enhanced substrate efficiency in many cases. The typical order of preference for 9-substituents was 2'-deoxyribofuranosyl greater than ribofuranosyl greater than arabinofuranosyl greater than H. Acyclic nucleosides (9-[(hydroxy-alkyloxy)methyl]purines) were usually more efficient substrates than were 2'-deoxyribonucleosides. The kinetic constants of a variety of purine analogs revealed that the pyrimidine portion of the purine ring system was a more important determinant of substrate activity than the imidazole portion. The efficient oxidation of a variety of nucleosides suggests that detoxification of naturally occurring nucleoside analogs might be an important aspect of the physiological role of this enzyme. Overall, the data presented serve as a guide for predicting the susceptibility of heterocycles to oxidation by this enzyme.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Effects of metabolic inhibitors on the release of glutamate from the retina

—The superfused, isolated retina of the chicken was used to investigate the mechanisms responsible for the increase in retinal transparency and the release of glutamate associated with stimuli known to elicit spreading depression (SD). We sought to distinguish between (1) mechanisms involving glutamate-induced increase in Na⁺ permeability and consequent uptake of extracellular material into the intracellular compartment and (2) mechanisms involving interference with operation of the Na⁺ pump that would result in a similar uptake of extracellular materials. Tetrodotoxin (which inhibits inward movements of Na⁺) depressed the transparency increase caused by stimulation with glutamate but not that elicited by application of KCl. Ouabain (which inhibits the Na⁺ pump) caused a marked increase in tissue transparency. The application of inhibitors of the aerobic metabolism, such as DNP or cyanide, or deprivation of O₂ had no effect on the retinal transparency; results suggesting that the energy for the Na⁺ pump could be supplied by glycolysis. Indeed iodoacetate (which inhibits glycolysis) caused a marked change in transparency. Furthermore we found evidence for a compound in the superfusion fluid supplemented with iodoacetate that may be a reaction product of glutamate and iodoacetate. In some preparations superfusion with glucose-free solutions caused a slowly developing increase in transparency and release of glutamate; in others the increase in transparency was more sudden and there was a larger release of glutamate. Seemingly, interference with the tissue metabolism can cause an uptake of extracellular material either by arrest of the Na⁺ pump or by the release of glutamate, depending on the conditions of the experiment.     

Sharpening of directional selectivity from neural output of rabbit retina

The estimation of motion direction from time varying retinal images is a fundamental task of visual systems. Neurons that selectively respond to directional visual motion are found in almost all species. In many of them already in the retina direction selective neurons signal their preferred direction of movement. Scientific evidences suggest that direction selectivity is carried from the retina to higher brain areas. Here we adopt a simple integrate-and-fire neuron model, inspired by recent work of Casti et al. (2008), to investigate how directional selectivity changes in cells postsynaptic to directional selective retinal ganglion cells (DSRGC). Our model analysis shows that directional selectivity in the postsynaptic cells increases over a wide parameter range. The degree of directional selectivity positively correlates with the probability of burst-like firing of presynaptic DSRGCs. Postsynaptic potentials summation and spike threshold act together as a temporal filter upon the input spike train. Prior to the intricacy of neural circuitry between retina and higher brain areas, we suggest that sharpening is a straightforward result of the intrinsic spiking pattern of the DSRGCs combined with the summation of excitatory postsynaptic potentials and the spike threshold in postsynaptic neurons.     

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of³H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.Special issue dedicated to Dr. Frederick E. Samson     

The functions of acetylcholine in the rabbit retina

Rabbit retinas were incubated in vitro under conditions known to maintain their physiological function. The acetylcholine stores of the cholinergic amacrine cells were labelled by incubation in the presence of [3H]choline. The tissue was then mounted in a fast-flow superfusion chamber, and the release of [3H]acetylcholine under various conditions was measured by liquid cation exchange or high-voltage electrophoresis. When the retina was stimulated by flashing light, the rate of appearance of radioactive acetylcholine in the superfusate increased, with a latency shorter than the resolution of the system. The rate of release of acetylcholine remained elevated as long as the light was flashing, and returned rapidly to baseline when the light was extinguished. A one minute stimulation with steady light caused a burst of acetylcholine release following stimulus onset and a second, smaller, burst following stimulus cessation. In the presence of 2-amino-4-phosphonobutyrate (APB), an agent known to eliminate selectively the transmission of ON responses to the proximal retina, steady light caused acetylcholine release only at stimulus cessation. Other retinas were labelled with [3H]choline, then incubated for 10-80 min in the presence of flashing light (to promote acetylcholine release) and either control medium or medium containing 100 micron APB (to prevent release from cells activated by stimulus onset). These retinas were quick-frozen, freeze-dried and radioautographed on dry emulsion. In retinas incubated under control conditions [3H]acetylcholine was initially present within two bands within the inner plexiform layer. The two bands became fainter together as the tissue's [3H]acetylcholine was released. APB selectively retarded the depletion of [3H]acetylcholine from the band nearest the ganglion cell layer. We conclude that the displaced cholinergic amacrine cells release acetylcholine at the transient when light appears, and the conventionally placed cholinergic amacrine cells release acetylcholine at the transient when light is extinguished. The retinal ganglion cells that receive a light-driven cholinergic input are distinguished from those that do not by a great sensitivity to slow stimulus motion. It is proposed that the dense plexus of cholinergic dendrites and the transient nature of acetylcholine release combine to create the local subunit that enables detection of motion within regions smaller than those ganglion cells' receptive fields.     

Autoradiographic identification of acetylcholine in the rabbit retina

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.     

Localization of GABAA receptors in the rabbit retina

The distribution of gamma-aminobutyric acid_A (GABA_A) receptors in the rabbit retina is investigated and compared with the distribution of GABAergic neurons using immunocytochemical methods. Antibodies against the 1, 2/3, and 2 subunits of the GABA_A receptor label subpopulations of bipolar, amacrine and ganglion cells. Double labeling experiments show that the 2 subunit is colocalized with the 1 and the 2/3 subunits in bipolar, amacrine and ganglion cells. Electron microscopy reveals that in the outer plexiform layer, GABA_A receptor immunoreactivity is present on dendrites of cone bipolar cells adjacent to the cone pedicles. Bipolar cell dendrites are also receptor-positive at synapses from interplexiform cells. Some receptor immunoreactivity is found intracellularly in processes of horizontal cells. In the inner plexiform layer, GABA_A receptor immunoreactivity is present on both rod bipolar and cone bipolar axon terminals at putative GABAergic input sites. Amacrine and ganglion cell processes in sublamina a and b are also labeled.     

Organotypic culture of adult rabbit retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.     

Potassium-stimulated release of GABA,glycine, and taurine from the chick retina

The effect of depolarizing potassium concentration on the release of [¹⁴C]glycine, [³H]GABA, and [³⁵S]taurine was investigated in the whole chick retina and in a synaptosomal fraction prepared from the chick retina. In the whole retina, increasing potassium concentration above 40 mM resulted in an increased release of the three amino acids. The release of glycine was the most stimulated and that of taurine, the least. The potassium-evoked release of glycine and GABA was calcium dependent. In the synaptosomal fraction, 68.5 mM potassium significantly stimulated the efflux of GABA and glycine by a calcium-dependent mechanism. The release of taurine from this fraction was unaffected by high potassium.     

Platelet-activating-factor-induced serum-albumin release from rabbit platelets in the absence of calcium

Using a radioimmunoassay for rabbit-serum albumin, platelet-activating-factor-induced serum-albumin release by platelets was monitored under non-aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet-activating-factor-aggregating effect. Serum-albumin release was much greater than serotonin release.