The dissociation of evoked release of [3H]-GABA and of endogenous GABA from chick retina in vitro

The release of prelabeled [³H]-GABA and endogenous GABA evoked by KCl and excitatory amino acid analogs was compared in intact chick retina and retina previously lesioned by the neurotoxins, kainic acid and N-methyl-d,l-aspartic acid. Significant discrepancies were found in the results obtained by the two techniques; and while changes in endogenous GABA release after neurotoxin lesions correlated with retinal glutamate decarboxylase activity, a marker for GABAergic neurons, the release of [³H]-GABA did not. These results call into question the validity of the prelabeling technique for studying GABA release from retina.     

The action of peptides on the mudpuppy electroretinogram (ERG)

Two neuropeptide substances were applied to the mudpuppy retina while the electroretinogram (ERG) was recorded. A low concentration of somatostatin (10(-9)M) was found to be a potent agent in increasing the amplitudes of all the oscillatory potentials (OPs) of the ERG. There was no appreciable change of the threshold sensitivity or the stimulus response curve of the b-wave. The highest concentration tested (10(-6)M) reduced the first OP to about the same amplitude as at the start of the experiment and attenuated the later OPs. A decrease of the suprathreshold b-wave was induced by the highest concentrations (10(-7), 10(-6)M) of somatostatin. A low concentration of substance P (10(-11)M) selectively and differentially decreased the earlier OPs (O1O2). Higher concentrations (10(-10), 10(-9)M) diminished the earlier OPs further, reduced the later OPs and decreased the supra-threshold a- and b-waves. The results support previous suggestions that the OPs reflect activities in feedback circuits initiated by the amacrines and indicate that somatostatin and substance P through separate mechanisms seem to interact with the inhibitory neuronal circuits which have been suggested to give rise to the OPs. Secondly, in agreement with previous work, the OPs appear to have a different origin from the b-wave. Thirdly, two separate classes of amacrines, each with a different transmitter, seem to be associated with different physiological roles.     

Autoradiographic localizations of [35S]-sulfate and [3H]-glucosamine in the hamster ciliary epithelium

The localization of [35S]-sulfate and [3H]-glucosamine in the hamster non-pigmented ciliary epithelium was studied by light and electron microscopic autoradiography. The radioactivity was concentrated in the Golgi complex of the non-pigmented ciliary epithelium 10 mins after injections of [35S]-sulfate or [3H]-glucosamine. Silver grains of both isotopes covered the cytoplasm 60 mins after the administration and were then associated with the basal infoldings. Radioactivity of [3H]-glucosamine was observed in the basal lamina of the non-pigmented ciliary epithelium and the ciliary zonule; however, the localization of [35S]-sulfate in the basal lamina and the ciliary zonule was not demonstrated. These data lend support to the idea that the non-pigmented ciliary epithelium may synthesize glycosaminoglycans (GAG) and secrete non-sulfated glycosaminoglycans into the posterior chamber.     

Retinal uptake and release of [3H]DABA

The uptake of [³H]DABA was studied in rat, guinea-pig and cat retinas in vivo and in rabbit retinas both in vivo and in vitro by autoradiography. [³H]DABA was preferentially accumulated by a type of amacrine cell but after brief incubation or shortly after intravitreal injections there was not only a neuronal uptake but also a glial one. In guinea-pigs the glial labelling in vivo was significant also after 4 hr.The glial uptake (rabbits) was found to be saturable and temperature dependent as expected for an active uptake mechanism. The K_m of DABA uptake was 2·11 × 10^?5m and V_max 2·38 × 10^?5 mol/mg/min. GABA competitively inhibited the DABA uptake. The uptake was not statistically significantly influenced by Ω-amino acids such as glycine, β-alanine, α-alanine or ?-aminocaproic acid.The effect of different stimuli on the spontaneous efflux of radioactivity from [³H]DABA preloaded rabbit retinas was studied with [³H]DABA localized to neurons. Light flashes evoked a small but not statistically significant increased release whereas 40 mm-K⁺ evoked an immediate and large increase. Unlabelled DABA, GABA and β-alanine (10^?5m) increased the spontaneous efflux of [³H]DABA but not glycine.It is concluded that there is in the retina of rats, guinea-pigs, cats and rabbits, a glial high affinity uptake mechanism in addition to the neuronal uptake. DABA seems to be transported by the same mechanism as GABA in both systems. The DABA seems to be better retained in neurons than in glia in rats, cats and rabbits, which allows it to be used as a neuronal marker.     

High affinity uptake sites for taurine in the retina

The uptake of [³H]taurine from 5 μm solutions by retinas from the rat, mouse, guinea pig, baboon, pigeon, cat and frog, has been studied with L.M. autoradiography. Labelling in photoreceptor cells and pigment epithelium are dominant features. There is also uptake into bipolar or amacrine neurones, or glial cells, which varies with species.     

Stereospecific [3H]naloxone binding associated with opiate receptors in bovine retina

Using the tritiated opiate antagonist [³H]naloxone, stereospecific opiate receptor binding was demonstrable in a bovine retina homogenate. Scatchard analysis of the saturation experiments revealed an apparent dissociation constant (K_D) of about 8·5 nm and a maximal number of binding sites (B_max) of about 0·24 pmol/mg protein. The properties of opiate receptor binding in bovine retina parallel those found in the rat brain. Agonist binding was altered by sodium ions. Subcellular distribution of stereospecific [³H]naloxone binding showed the highest density of binding sites in the P₁-fraction and high concentrations in the P₀- and P₂-fraction.     

5-hydroxytryptamine: its facilitative action on [3H]dopamine release from the retina

5-Hydroxytryptamine (5-HT) at 5 X 10(-4) M caused a two-fold increase in [3H]dopamine (DA) release from retinal particulate fractions of the carp (Cyprinus carpio). The 5-HT action was dose- and Ca2+-dependent. The three 5-HT agonists examined (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine and tryptamine) were more effective than 5-HT on [3H]DA release. 5,6-Dihydroxytryptamine was the strongest competitor among drugs tested for [3H ]DA uptake, but did not evoke any significant release of [3H]DA. Noradrenaline (or DA) at 5 X 10(-4) M produced a large increase in [3H]DA release from the particulate fractions, but its action was Ca2+-independent. The 5-HT-induced DA release could be seen in the frog retina but not in the rat retina, which does not contain indoleamine-accumulating cells. The results obtained strongly suggest that 5-HT stimulates [3H ]DA release through a receptor mechanism on DAergic terminals and modifies the DA-cell's function in the retina.     

D-[3H]-galactose incorporation in the bovine retina: specific uptake and transport of the radiolabel in cones

When bovine neural retinas are incubated in Krebs-Henseleit buffer with D-[3H]-galactose, autoradiography reveals that there is a rapid uptake of the tritium label into the inner segments of cones, but not of rods. Pulse--chase studies show that the label is first associated with the Golgi apparatus in the cones, then appears to travel around the nucleus and along the cone fibre (homologous to an axon) to the synaptic pedicle. The cone-specific label travels along the fibre at a rate of about 0.5-1.0 mm per day. Label is also found in endothelial cells and Müller cells, but does not persist in the Müller cells as long as in the cones. The striking difference between rod and cone labelling may reflect fundamental differences in the neurochemistry of these two photoreceptor cell types.     

The action of inhibitory neurotransmitters, γ-aminobutyric acid and glycine may distinguish between the area centralis and the peripheral retina in cats

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine, and of their antagonists, bicuculline and strychnine, were compared between ganglion cells from the central and peripheral retinae of optically intact eyes in barbiturate-anaesthetised cats. The visual response of on-cells was inhibited by GABA and enhanced by bicuculline. The visual response of off-cells was inhibited by glycine and enhanced by strychnine. The sensitivity of cells to the transmitters was lower in the peripheral retina than in the area centralis, whilst the sensitivity to the antagonists was similar in both regions of the retina. Cells from the area centralis were inhibited by either GABA or glycine, but never both. Cells from the periphery were less selective and were inhibited by both transmitters.     

Taurine uptake processes in the isolated rabbit retina and the effects of light

As part of a study on the mechanisms involved in regulating photoreceptor taurine levels, we have examined the characteristics of [3H]-taurine uptake by the isolated rabbit retina. The effects of light on taurine accumulation have also been studied. Rabbit retinas quickly accumulated [3H]-taurine and tissue: medium ratios of 50:1 were attained after 60 min incubation at 37 degrees C. Under these conditions no metabolites of [3H]-taurine were detected in the tissue. The efflux of [3H]-taurine from the retina was extremely slow, less than 5% of the accumulated radioactivity being released in a 30 min incubation in fresh medium. Thus, in subsequent experiments the accumulation of radioactivity was taken as a measure of [3H]-taurine uptake. Non-linear regression analysis of kinetic data revealed that taurine was accumulated by separate high- and low-affinity transport processes, the kinetic parameters being Kmh = 93 +/- 12 microM; Vmh = 72 +/- 7; KmL = 8.8 +/- 5 mM; VML = 274 +/- 79 nmol min-1 g-1 wet weight respectively. The properties of the high- and low-affinity taurine uptake processes were very similar. Both were temperature sensitive, particularly between 25 and 37 degrees C and sodium- and chloride-dependent, and were inhibited by metabolic inhibitors. The substrate specificities of the high- and low-affinity uptake processes were also similar, both processes being inhibited by beta-alanine, guanidinoethylsulphonate (GES) and GABA, but not by alpha-alanine or glycine. Hypotaurine selectively inhibited the high-affinity uptake process for [3H]-taurine. Exposure of retinas to continuous light did not affect either the high- or the low-affinity uptake of [3H]-taurine compared with dark-adapted controls. However, flickering light (0.5-30 Hz, 25% duty cycle) reduced the high-affinity accumulation of [3H]-taurine by as much as 50%. The reduction in [3H]-taurine may be due to a localized decrease in uptake (or possibly an increased release) by the photoreceptors because the same reduction was found when synaptic transmission in the retina was blocked by exposure to medium containing high Mg/low Ca. High Mg/low Ca did not itself affect taurine accumulation.     

Subcellular distribution of [3H]choline,choline acetyltransferase,acetylcholinesterase and butyrylcholinesterase activities in rabbit retina

In order to obtain further information on the retinal cholinergic system, the uptake and subcellular distribution of [³H]choline by the rabbit retina in vitro was examined. Low (6·5 × 10^?8m) concentrations of choline were used in order to study the high affinity uptake system which appears to be exclusively associated with cholinergic neurotransmitter function in the mature CNS.The subcellular distribution of enzymes of the retinal cholinergic system (choline acetyl-transferase, ChAT; acetylcholinesterase, AChE; and butyrylcholinesterase, BuChE) were also studied and compared with the subcellular distribution of [³H]choline.Rabbit retinae rapidly accumulated radioactivity from the external medium and after 45 min incubation, tissue/medium ratios of approximately six were obtained. A preliminary investigation demonstrated that the uptake was temperature dependent and was reduced by about 50% in the absence of sodium ions or the presence of hemicholinium (H.C.3.) (0·1 mm) in the incubation medium. Primary subcellular fractionation of retinae incubated with [³H]choline indicated that accumulation of radioactivity by the crude mitochondrial fraction (P2) was reduced if sodium was absent from the incubations. Since the P2 fraction from rabbit retina contains synaptosomes, this result may be indicative of a sodium-dependent high-affinity accumulation of exogenous choline by retinal cholinergic synaptosomes.On continuous, sucrose density gradients more than 80% of the P2 radioactivity appeared to be localized in a population of osmotically-sensitive particles which had a median equilibrium density equivalent to 1·25 m-sucrose. Furthermore, over 85% of the ChAT activity on such gradients was present in particles with the same equilibrium density as those accumulating choline.The primary subcellular distribution of AChE and BuChE showed that both enzymes were present in the total particulate fractions (P1 and P2) although BuChE was a relatively more soluble enzyme than AChE. Both enzymes had a broader distribution than ChAT on density gradients. The likely localization of AChE and BuChE in synaptosome membrane fragments and glial particles respectively may thus point to the ubiquitous nature of such particles on sucrose gradients. The considerable activity of the cholinergic enzymes and the large proportion of [³H]choline accumulated by the crude nuclear fraction (P1) may be due to the presence of the large photoreceptor synaptosomes present in this fraction, but more likely to the presence of unbroken tissue fragments.It is concluded that the rabbit retina possesses a sodium-dependent, high affinity uptake process for choline. This process is present in particles which may prove to be retinal cholinergic nerve endings.     

The effect of light and potassium depolarization on the release of endogenous amino acids from the isolated rat retina

The release of endogenous amino acids from the isolated rat retina was studied. A consistent pattern of spontaneous resting release was found with taurine being released at the fastest rate ($\text{8·9 nmol}\text{10 min}$) and GABA the lowest ($\text{0·7 nmol}\text{10 min}$). Exposure of retinae to medium containing high concentrations of potassium produced calcium-dependent increases in the efflux of taurine and glycine but not of GABA, alanine, glutamate or glutamine. Photic stimulation of dark-adapted retinae had no effect on the efflux of any amino acid.     

GABA and glycine effects on the bipolar cells of the carp retina

Effects of GABA and glycine applied electrophoretically were examined on bipolar cells in the carp retina. When applied at the outer plexiform layer, GABA produced a small depolarization in on-center cells, but a small hyperpolarization in off-center cells. These effects were concluded to be mediated by photoreceptor cells. When applied at the inner plexiform layer, GABA produced a hyperpolarization in on-center cells but no response in off-center cells. This response was resistant to Co2+ ions. Thus GABA is very like the inhibitory neurotransmitter from amacrine to on-center bipolar cells. Glycine, when applied in either layer, did not have a noticeable effect on either type of bipolar cells.