Immunohistochemical characterization of the chick marginal retina

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Endo-oligopeptidase A, a putative enkephalin-generating enzyme, in the vertebrate retina.

Endo-oligopeptidase A, EC 3.4.22.19, converts small enkephalin-containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo-oligopeptidase. A found in pigeon retinae (30.3 +/- 7.3 mU/mg, mean +/- standard deviation) was four times higher than in rabbit retinae (7.0 +/- 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5,5'-dithiobis(2-nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo-oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double-labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo-oligopeptidase A-like immunoreactivity also contained [Leu5]-enkephalin-like immunoreactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo-oligopeptidase A in enkephalin generation in vivo.

Efferent projections of the pigeon intergeniculate leaflet containing neuropeptide Y

The present study was performed to map efferent projections of the pigeon intergeniculate leaflet (IGL) to other visual structures, with emphasis on the pathways containing neuropeptide Y (NPY). After injections of an anterograde tracer (Phaseolus vulgaris leucoagglutinin) into the IGL, labeled axons and presumptive terminals were seen in several retinorecipient and visually-related nuclei. All such areas contained immunoreactive fibers to antibodies against NPY. Electrolytic lesion of the IGL provoked a marked reduction in the number of NPY-labeled fibers in these visual structures. The data suggest that the IGL is the source of NPY-labeles axons which occur in many visual nuclei of the pigeon brain

Neurotransmitters, receptors, and neuropeptides in the accessory optic system: an immunohistochemical survey in the pigeon (Columba livia).

Immunohistochemical techniques were used to survey the distribution of several conventional transmitters, receptors, and neuropeptides in the pigeon nucleus of the basal optic root (nBOR), a component of the accessory optic system. Amongst the conventional neurotransmitters/modulators, the most intense labeling of fibers/terminals within the nBOR was obtained with antisera directed against glutamic acid decarboxylase (GAD) and serotonin (5-HT). Moderately dense fiber plexuses were seen to label with antibodies directed against tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). GAD-like immunoreactivity (GAD-LI) was found in many small and medium-sized perikarya within the nBOR. Some of the medium-sized cells were occasionally positive for ChAT-LI. Cell body and dendritic staining was also commonly seen with the two tested antisera against receptors-anti-GABA-A receptor and anti-nicotinic acetylcholine receptor. The antisera directed against various neuropeptides produced only fiber labeling within the nBOR. The densest fiber plexus staining was observed with antiserum against neuropeptide Y (NPY-LI), while intermediate fiber densities were seen for substance P (SP-LI) and cholecystokinin (CCK-LI). A few varicose fibers were labeled with antisera against neurotensin (NT), leucine-enkephalin (L-ENK), and the vasoactive intestinal polypeptide (VIP). Unilateral enucleation produced an almost complete elimination of TH-LI in the contralateral nBOR. SP-LI and CCK-LI were also decreased after enucleation. No apparent changes were seen for all other substances. These results indicate that a wide variety of chemically-specific systems arborize within the nBOR. Three of the immunohistochemically defined fiber systems (TH-LI, SP-LI, and CCK-LI fibers) were reduced after removal of the retina, which may indicate the presence of these substances in retinal ganglion cells. In contrast, the fibers exhibiting ChAT-LI, GAD-LI, 5-HT-LI, NPY-LI, NT-LI, L-ENK-LI, and VIP-LI appear to be of nonretinal origin. Two different populations of nBOR neurons exhibited GAD-LI and ChAT-LI. However, these two populations together constituted only about 20% of the nBOR neurons.

Telencephalic and pretectal modulation of the directional selectivity of accessory optic neurons in the pigeon

Direction-selective units within the accessory optic system of the pigeon were shown to respond more strongly to motion along two main directions, downward-nasal and upward-temporal. Following ipsilateral telencephalic or pretectal lesions, these directions were modified in a systematic way. In the former, the principal response directions were downward-nasal and temporal and in the latter, downward-temporal and upward-temporal. These data indicate that the non-retinal afferents play an important role in the functional organization of the accessory optic system