Distribution of GABA immunoreactivity in the cat retina: a light- and electron-microscopic study

The distribution of GABA-like immunoreactivity in the cat retina was studied through the use of preembedding immunocytochemistry for light microscopy and by postembedding immunogold techniques for electron microscopy. Staining was observed in both inner and outer plexiform layers. Approximately 30% of the somata in the amacrine portion of the inner nuclear layer were immunoreactive and included amacrine and interplexiform cells. Horizontal cells and a subpopulation of cone bipolar cells were also stained. In the ganglion cell layer, staining was observed in both small- and medium-sized neurons. GABA-labeled amacrine cells were presynaptic to somata of amacrine cells and to dendrites of amacrine, bipolar, and ganglion cells. Bipolar cells were a major target, receiving more than 60% of all labeled synapses in the inner plexiform layer. Many of these contacts were reciprocal synapses. These findings support a major role for GABA-labeled amacrines in providing feedback inhibition to bipolar cells in the inner retina.     

GABA(A) receptor immunoreactivity is transiently expressed in the developing outer retina

Extensive evidence has suggested a trophic role of gamma-aminobutyric acid (GABA) on developing cone photoreceptors in postnatal retina. In a previous study, we showed that GABA raises intracellular calcium levels in the developing cones via activation of GABA(A) receptors. Using confocal microscopy in conjunction with immunocytochemistry, we have now demonstrated that (1) GABA(A) receptor subunits are localized on cone cell bodies as well as on cone pedicles, indicating that GABA has a direct, rather than indirect, effect on cones and (2) the temporal expression of GABA(A) receptor subunits coincides with the developmental effects of GABA on cone synaptogenesis. An antibody against the beta 2/3 subunits of the GABA(A) receptor and a specific cone marker peanut-agglutinin lectin (PNA) were used to double-label wholemount neonatal retinal preparations. Results show that GABA(A) receptors are transiently expressed on cone photoreceptors in the early stages of postnatal retinal development. GABA (A)receptor immunoreactivity is clearly present on cone cell bodies and their processes and on other--as yet unidentified--elements (horizontal cells?) in the outer plexiform layer. Immunoreactivity decreases within cone photoreceptor somata after postnatal day 5, but persists in the processes of the outer plexiform layer until day 7. Our results provide support for the hypothesis that GABA acts as an important developmental regulator of cone photoreceptor maturation.     

Calcium binding protein immunoreactivity in pigeon retina

Pigeon retina has been mapped immunocytochemically for vitamin D-dependent calcium-binding protein (D-CaBP). Immunoreactivity was found in the cones of the yellow field, but not in photoreceptors of the red field. The D-CaBP-containing cones were a subpopulation of those in the yellow field having straight fibres leading to their synaptic terminals. D-CaBP immunoreactivity was also found in horizontal cells, the amount present varying according to position along the retina, and in some amacrine cells. Immunoblots of pigeon retinal proteins separated by SDS-polyacrylamide gel electrophoresis indicated two D-CaBP forms, having apparent molecular weights of 27000 and 29000. Both these forms of D-CaBP have been found previously in rat and pigeon brain.     

Post-natal development of TH-like immunoreactivity in the rat retina

The post-natal development of catecholaminergic neurons has been studied in the rat retina, using tyrosine-hydroxylase immunohistochemistry. The first TH-like immunoreactive cells were observed by the third post-natal day. The differentiation of neurons and the development of their processes continued until and after, the opening of the eyes (14-15th post-natal day). The catecholaminergic system was fully developed by three weeks of age. The use of retinal flat mounts treated with PAP-immunoperoxidase technique allowed a morphological observation of TH-like immunoreactive whole neurons.     

Neuropeptide Y-like immunoreactivity in neurons of the human retina

The distribution of neuropeptide Y-like immunoreactivity (NPY-LI) was investigated in wholemounts and in transverse sections of the human retina. NPY-LI was localized to the soma and axonal processes of large ganglion cells (GCs) and to the soma and dendritic arborization of amacrine cells (ACs). NPY-LI GCs were unevenly distributed across the retina, the highest density of 875 cells/mm2 was found in the fovea centralis and the lowest density of 15 cells/mm2 in the peripheral retina. The total number of NPY-LI GCs in the retina was estimated to be about 85,000. The soma sizes of NPY-LI GCs increased from 116 microns 2 +/- 23 (s.d.) in the retinal centre to 251 microns 2 +/- 57 in the retinal periphery. The soma size of NPY-LI ACs was in the range of 40 and 50 microns 2. In transverse sections NPY-LI was seen to be localized to the optic fibre layer, to the somata of GCs, to the scleral sublamina of the inner plexiform layer (AC dendrites) and to the innermost part of the inner nuclear layer (somata of ACs). The gradients of soma sizes and retinal distribution of NPY-LI GCs were taken as an indication that they correspond to the class of large to very large GCs, previously identified in the human retina by Golgi impregnation.     

GABA receptors on horizontal cells in the goldfish retina

We investigated the localization of GABA(A) and GABA(C) receptors in horizontal cells (HCs) and HC axon terminals (ATs) dissociated from goldfish retina, using whole-cell patch-clamping recordings. Applications of GABA on HCs induced two groups with inward currents at the holding potential of -50 mV: One was a sustained inward current in the H1 cell, with one type of HCAT (AT1), and the other was a transient inward current in other HC soma and HCAT (AT2). Co-application of GABA with bicuculline or SR95531, GABA(A) receptor antagonists, showed a non-blocking effect in the sustained current, but a blocking effect in the transient current. The sustained current was evoked by cis-4-aminocrotonic acid (CACA), a GABA(C) receptor agonist, while the transient current was not induced by CACA, but mimicked by muscimol, a GABA(A) receptor agonist. Both the sustained and transient currents were completely blocked by picrotoxin and not mimicked by baclofen, a GABA(B) receptor agonist. Thus H1 cell and AT1 have GABA(C) receptors, while H2, H3 cells and AT2 have GABA(A) receptors.     

A light-driven rhythm in neurotensin-like immunoreactivity in the chicken retina

Purpose: To determine if the pattern of release of neurotensin from the enkephalin-, neurotensin- and somatostatin-like immunoreactive amacrine cells in response to light and dark is the same as that of the enkephalins and somatostatin. Methods/Results: Both the enkephalins and somatostatin are released at high rates in the dark and at lower rates in the light, and these rate changes are reflected in increasing intracellular levels of the peptides in vivo in the light and decreasing levels in the dark The levels of neurotensin-like immunoreactivity show a similar diurnal light-driven and non-circadian rhythm in vivo. Conclusion: This implies that the actual release rates of neurotensin follow the same patterns as those demonstrated in vitro for the enkephalins and somatostatin.     

Characterization of neuropeptide Y-like immunoreactivity in vertebrate retina

We have determined the endogenous levels of neuropeptide Y-like immunoreactivity (NPY-LI) in retinal extracts from pigs, cats, rabbits, chickens, and frogs by radioimmunoassay (RIA). The NPY-LI levels varied among the species. The highest concentration was found in frog retina, where seasonal variations were seen, 861 +/- 31 pmol g(-1) wet weight in the autumn and 334 +/- 26 pmol g(-1) wet weight in the spring. Lower levels were demonstrated in chicken and pig retina, 4.1 +/- 0.4 and 3.6 +/- 0.3 pmol g(-1) wet weight, respectively. The lowest concentration was demonstrated in rabbit retina, 2.0 +/- 0.3 pmol g(-1) wet weight. (All values are expressed as mean +/- S.E.M.). The NPY-LI in pig, rabbit, chicken and frog retina was characterized by high-performance liquid chromatography (HPLC). The main part of the extracted NPY-LI had an elution volume close to that to the porcine NPY. We have also analysed the evoked release of endogenous NPY from frog retina, induced either with light flashes (3 Hz, 300 lx), or with potassium depolarization of the neurons (40 mM). Light flashes and potassium induced an increased release of NPY-LI of 61 and 77%, respectively. NPY-LI in the efflux had the same HPLC retention time as that extracted directly from the retina.     

HIOMT-like immunoreactivity in the vertebrate retina: a species comparison

Localization of the melatonin-synthesizing enzyme, hydroxyindole-O-methyltransferase (HIOMT)-like immunoreactivity was examined by light and electron microscopic immunocytochemistry, in the retinas of several species that have been used as animal models to study the retinal melatonin system. HIOMT-like immunoreactivity was observed in the retinal photoreceptors of rabbit, pigmented rat, guinea-pig, chick, goldfish. African clawed toad, and leopard frog. Additionally, most species displayed HIOMT immunoreactivity in a population of bipolar cells in the inner nuclear layer. At the ultrastructural level, HIOMT-like immunoreactivity was localized to the cytoplasm of rod and cone photoreceptors, and a population of cone bipolar cells. These observations are identical to earlier observations in the human retina. The similar pattern of HIOMT-like immunoreactivity among species suggests a phylogenetic conservation of the melatonin-synthesizing capability of retinal photoreceptors and some bipolar cells.     

GABA-like immunoreactivity in the vertebrate retina: a species comparison

Rabbit antisera directed against gamma-amino butyric acid (GABA) conjugated to bovine serum albumin was used to localize neurons containing GABA-like immunoreactivity in the retinas of nine species of animals: human, cat, rabbit, rat, chicken, turtle, frog, mudpuppy and goldfish. The retinas of all species contained GABA-like labeling in several populations of amacrine cells in the inner nuclear layer, cells in the ganglion-cell layer that may include displaced amacrine cells and in fibers in the inner plexiform layer and in the optic nerve fiber layer. Labeled horizontal cells were found in cat and in all non-mammalian retinas. Labeled interplexiform cells were found in rat, rabbit, cat and human retinas. Labeled bipolar cells were restricted to frog and mudpuppy retinas. The distribution of anti-GABA is usually similar to that of anti-glutamic acid decarboxylase and neuronal [3H]GABA uptake, indicating good correspondence between these 'GABAergic' markers. However, several significant differences among these markers are discussed.     

Localization of substance P-like immunoreactivity within the monkey retina

Substance P (SP)--like immunoreactivity has been localized to distinct retinal cell populations in the primate Macaca nemestrina by means of immunohistochemical techniques with a well-characterized monoclonal antiserum directed to SP. The specificity of the immunoreactive staining was established by absorption of the antiserum with 10 micro M synthetic SP. Specific SP-like immunoreactivity was observed within varicose processes located in the outer plexiform layer, inner nuclear layer (INL), and in three bands within the inner plexiform layer (IPL). SP-immunoreactive somata were located in the proximal INL, IPL, and ganglion cell layer. These studies have thus identified SP-containing amacrine cells, interstitial amacrine cells, and displayed amacrine cells. In addition, these studies suggest the existence of SP-containing interplexiform cells and perhaps ganglion cells.     

Vitamin D-dependent calcium binding protein immunoreactivity in human retina

Using immunohistochemistry, vitamin D-dependent calcium binding protein (D-CaBP) has been detected in human retina. In the photoreceptor layer the cones are positive but the rods are negative. In the inner nuclear layer, horizontal cells and some bipolar cells are D-CaBP. In the ganglion cell layer both small and large somata are immunoreactive for D-CaBP. Beaded fibres from the outer plexiform, inner plexiform and fibre layers are also positive.     

Double-label analysis of GAD- and GABA-like immunoreactivity in the rabbit retina

Rabbits were injected intravitreally with colchicine and the retinas were subsequently processed for simultaneous visualization of GAD-like and GABA-like immunoreactivity (LIR) at the light microscopical level. The use of specific antisera from unrelated species minimized the possibility of crossreactivity between the two markers. Colocalization of GABA-LIR and GAD-LIR was observed in approximately 32% of somata on the inner border of the inner nuclear layer (INL) and roughly 15% of the somata in the ganglion cell layer (GCL). All GAD-positive cells were double labeled whereas 15% of the GABA-LIR cells in the INL and 52% of those in the GCL were negative for GAD-LIR. Both markers were seen throughout the inner plexiform layer. In addition, processes coursing through the OPL, perhaps from interplexiform cells, and horizontal cell bodies were double-labeled with these GABAergic probes.     

Subcellular distribution of GABA receptors in bovine retina.

Two synaptosomal fractions were isolated from bovine retina and analyzed for GABA receptors using a [³H]GABA binding assay. Two receptor sites were identified: one with an affinity constant of 345 nm, which was uniformly distributed between the two fractions, and a second site with an affinity constant of 38 nm, which was primarily associated with the synaptosomal fraction enriched in photoreceptor cell terminals. Both sites demonstrated a high degree of pharmacologic specificity. The GABA receptor agonist, muscimol, was a more potent displacer than imidazolacetic acid or bicuculline; uptake blockers, diaminobutyric acid and β-alanine were poor displacers. These data provide further support for the role of GABA as a neurotransmitter in retina. It also suggests the presence of a significant GABAnergic system in the outer plexiform layer of the bovine retina.     

Substance P immunoreactivity in normal human retina and in retinoblastoma

Substance P (SP) immunoreactivity was demonstrated using the indirect immunofluorescence technique in one normal and one retinoblastomatous human retina. In the normal retina SP immunoreaction was located in nerve fibres but not in the neurons in the inner plexiform layer. A similar location was observed in the histologically normal areas of the retinoblastoma sample. SP immunoreactive neurons, probably amacrine cells, were, however, observed in the transitional area between the normal retina and the tumour. The tumour mass, although mainly SP negative, contained clusters of pleomorphic cells with an intense SP immunoreaction. The general distribution of SP immunoreaction in human retina resembles that of other mammals. The positive SP immunoreaction in retinoblastoma cells suggests that the tumour either may have its origin in the amacrine cells or that the retinoblasts are capable of redifferentiating in the direction of the amacrine cell population. The general problems concerning the origin and pathogenesis of retinoblastoma are discussed.     

GABA agonists fail to protect the retina from ischemia-reperfusion injury

The purpose of this study was to test the hypothesis that ischemia/reperfusion injury in the rat retina may be ameliorated by reducing retinal metabolism with either hypothermia or inhibitory GABA agonists. The intraocular pressure of each right eye in rats was raised to 130 mmHg for 60 min with the left eye serving as normal control. The rats were divided into four groups in terms of drug and hypothermia treatment: (1) Untreated ischemia, (2) Hypothermia, (3) Baclofen/midazolam and (4) Baclofen/muscimol. Electroretinogram was recorded before ischemia and again after 10-day reperfusion. Histological analysis with H&E staining and cell counts was performed. Untreated ischemia/reperfusion resulted in severely reduced ERG responses. The ERG b-wave was reduced from 423 ± 144 μV to 130 ± 91 μV (mean ± SD, n = 5). With hypothermia the ERG b-wave was reduced from 499 ± 80 μV to 237 ± 111 μV (n = 4). With combinations of baclofen and midazolam the ERG b-wave was reduced from 432 ± 96 μV to 104 ± 67 μV (n = 7). In baclofen/muscimol treated eyes the ERG b-wave went from 426 ± 101 μV to 148 ± 118 μV (n = 6). The histological tissue damage was severe in untreated ischemia and the baclofen/midazolam and baclofen/muscimol groups, but less severe in the hypothermia group. The GABA agonists do not provide any protection in our ischemia/reperfusion model. Our results are consistent with earlier reports that hypothermia may be helpful in ischemic conditions in the retina.     

Amino acid immunoreactivity in normal human retina and after brachytherapy

We localised amino acids in the mid‐peripheral aged human retina and a retina that had undergone radiation treatment 10 years earlier. The distribution pattern of glutamate, γ‐amino butyric acid (GABA), glycine, glutamine and taurine, reflected patterns established in the primate retina. The retina that had undergone radiation exposure displayed both anatomical and neurochemical remodelling. The proximal retina comprised around 40 to 45 per cent of the total retina and neuronal kinesis and aberrant neuronal projections were also present. Amino acid neurochemistry was strikingly different with Müller cells displaying GABA loading, glycinergic neurons displaced and displaying a very high level of glycine labelling. We conclude that radiation exposure triggered these changes in the human retina and likely reflects general remodelling of structure and function following ischaemic damage to endothelial cells.