Connexin36, an essential element in the rod pathway, is highly expressed in the essentially rodless retina of Gallus gallus.

Electrical coupling provided by connexins (Cx) in gap junctions (GJ) plays important roles in both the developing and the mature retina. In mammalian nocturnal species, Cx36 is an essential component in the rod pathway, the retinal circuit specialized for night, scotopic vision. Here, we report the expression of Cx36 in a species (Gallus gallus) that phylogenetic development endows with an essentially rodless retina. Cx36 gene is very highly expressed in comparison with other Cxs previously described in the adult retina, such as Cx43, Cx45, and Cx50. Moreover, real-time PCR, Western blot, and immunofluorescence all revealed that Cx36 expression massively increased over time during development. We thoroughly examined Cx36 in the inner and outer plexiform layers, where this protein was particularly abundant. Cx36 was observed mainly in the off sublamina of the inner plexiform layer rather than in the on sublamina previously described in the mammalian retina. In addition, Cx36 colocalized with specific cell markers, revealing the expression of this protein in distinct amacrine cells. To investigate further the involvement of Cx36 in visual processing, we examined its functional regulation in retinas from dark-adapted animals. Light deprivation markedly up-regulates Cx36 gene expression in the retina, resulting in an increased accumulation of the protein within and between cone synaptic terminals. In summary, the developmental regulation of Cx36 expression results in particular circuitry-related roles in the chick retina. Moreover, this study demonstrated that Cx36 onto- and phylogenesis in the vertebrate retina simultaneously exhibit similarities and particularities.

Differential expression of connexins during histogenesis of the chick retina.

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.

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.

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.

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 microg/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 microg/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 microg/g) and 845 +/- 82 cells/mm2 (6 microg/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 microg/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.

A subpopulation of displaced ganglion cells of the pigeon retina exhibits substance P-like immunoreactivity.

Immunohistochemical and retrograde tracing techniques were combined to demonstrate the occurrence of displaced ganglion cells (DGCs) exhibiting substance P-like immunoreactivity (SP-LI) in the pigeon retina. Following injections of rhodamine-labeled latex microspheres into the nucleus of the basal optic root (accessory optic system), about 5200 DGCs were observed to contain rhodamine fluorescence in the contralateral retina. Approximately 26% of the retrogradely labeled DGCs also contained SP-LI. The soma sizes of the doubly labeled DGCs ranged from 12 to 24 microns, and their distribution mirrored the overall distribution of DGCs projecting to the nucleus of the basal optic root. The density of doubly labeled DGCs ranged from 2 to 15 cells/mm2, with density peaks occurring in the superior-nasal and inferior-temporal retinal quadrants. Larger DGCs projecting to the nBOR (25-32 microns) were never seen to contain SP-LI. Together with previous results of enucleation experiments, these data indicate the existence of a subpopulation of SP-LI DGCs which are connected with the accessory optic system in the pigeon. The present results also contribute information on the heterogeneity of retinal ganglion cells transmitters and modulators.

Thalamic origin of neuropeptide Y innervation of the accessory optic nucleus of the pigeon (Columba livia).

Immunohistochemical and tracing techniques were used in combination to reveal the source of a neuropeptide Y-like immunoreactive (NPY-LI) plexus in the nucleus of the basal optic root (nBOR) of the pigeon accessory optic system. Injections of rhodamine-labeled latex microspheres into nBOR produced retrograde labeling of a population of neurons interposed between the principal optic nucleus of the dorsolateral thalamus (equivalent to the mammalian dorsal lateral geniculate nucleus) and the ventral lateral geniculate nucleus. The retrogradely labeled neurons were distributed mainly in the immediate vicinity of the lateral, dorsal, and ventral aspects of the nucleus rotundus. Immunohistochemical methods revealed many NPY-containing somata within the same intergeniculate thalamic area. Double-labeling immunohistochemical and retrograde tracing experiments evidenced that many NPY-LI neurons in the intergeniculate area contained rhodamine microspheres that had been previously injected into the ipsilateral nBOR. The projection of that general thalamic area to the nBOR was then confirmed by means of anterograde transport of Phaseolus vulgaris leucoagglutinin. In these experiments, the intergeniculate region was demonstrated to project to all divisions of the nBOR and to every other retino-recipient structure, including the suprachiasmatic nucleus. Finally, electrolytic lesions of the intergeniculate area produced a dramatic reduction in the number of NPY-LI axons and terminals within the ipsilateral nBOR and also within other retino-recipient structures. These data indicate the existence of a thalamic NPY-LI projection to the pigeon nBOR of the accessory optic system. This chemically specific projection originates from the intergeniculate area, which was shown in this study to project to all other retino-recipient structures. Thus, NPY may have a role in the functional organization of the accessory optic system and also of the avian visual system as a whole.

Visual telencephalon modulates directional selectivity of accessory optic neurons in pigeons.

The directional selectivity of units within the nucleus of the basal optic root (nBOR) of the accessory optic system (AOS) was studied before and after lesions of the visual telencephalon (visual Wulst) in urethane-anesthetized pigeons. In intact pigeons, most nBOR units preferred upward motion with a temporal component or downward motion with a nasal component. The ipsilateral and bilateral telencephalic lesions generated a dramatic reduction in the number of cells with optimal responses to upward motion. The overall distribution of preferred directions was still bimodal following ipsilateral or bilateral Wulst lesions, with most units showing best responses to a straight temporal or to downward-nasal directions. The contralateral Wulst lesions produced, instead, a marked reduction in downward preferences. The nBOR units which were studied in these cases showed mainly upward-temporal and upward-nasal responses. These data suggest an involvement of the visual Wulst in the determination of the directional selectivity of nBOR neurons in the pigeon. Specifically, the responses of nBOR units to upward motion appeared to depend on the integrity of the telencephalic descending systems which impinge, in both direct and indirect ways, upon that AOS nucleus. Taken together with data for the mammalian AOS, the present results indicate that nonretinal afferents to AOS nuclei have an important role in the functional organization of that subcortical visual pathway.

Efferent projections of the pigeon intergeniculate leaflet containing neuropeptide Y.

The present study was preformed 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-labeled axons which occur in many visual nuclei of the pigeon brain.

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.

Chemically specific retinal ganglion cells collateralize to the pars ventralis of the lateral geniculate nucleus and optic tectum in the pigeon (Columba livia).

Immunohistochemical and retrograde tracing techniques were combined to study the retinal ganglion cells which project to the pars ventralis of the lateral geniculate nucleus (GLv) in the pigeon. Using two different fluorescent tracers, two histochemically-distinct populations of ganglion cells were found to project to both the GLv and the optic tectum. The first population of ganglion cells exhibited tyrosine hydroxylase-like immunoreactivity and represented about 20% of all ganglion cells which were retrogradely labeled from the GLv. The second population of ganglion cells showed substance P-like immunoreactivity and represented about 13% of all ganglion cells projecting to the GLv. These results confirm earlier suggestions that the retinal axons projecting to the GLv also project elsewhere and demonstrate that heterogeneity of retinal ganglion cells transmitters is evident even within a single retino-recipient nucleus such as the GLv.

Catecholaminergic subpopulation of retinal displaced ganglion cells projects to the accessory optic nucleus in the pigeon (Columba livia).

In birds, displaced ganglion cells (DGCs) constitute the exclusive source of retinal input to the nucleus of the basal optic root (nBOR) of the accessory optic system. Tyrosine-hydroxylase (TH) immunoreactivity was examined in the pigeon retina after injections of rhodamine-labeled microspheres into the nBOR. A population of about 400 DGCs was observed in each case to exhibit both TH immunoreactivity and rhodamine bead fluorescence. This corresponded to about 10-15% of the total number of identified DGCs in each retina. Double-labeled cells were medium- to large-size (12 to 20 microns in the largest axis) and were always located at the border between the inner nuclear and the inner plexiform layers. Their dendrites could be followed horizontally in lamina 1 of the inner plexiform layer for up to 300 microns from the cell body. The distribution of double-labeled DGCs appeared to be mostly peripheral, matching the overall distribution of identified DGCs. Larger DGCs (21-28 microns) were never seen to contain TH immunoreactivity. Examination of brain sections revealed plexuses of thin varicose TH-positive axons in all subdivisions of the nBOR. Unilateral enucleation produced an almost complete elimination of TH immunoreactivity in the contralateral nucleus. Such results suggest the existence of a population of catecholaminergic DGCs projecting into the accessory optic system of the pigeon. They also support the emerging hypothesis concerning the neurotransmitter heterogeneity of ganglion cells in the vertebrate retina.

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

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.

Interocular transfer of habituation in pigeons: mediation by tectal and/or posterior commissures.

Interocular transfer of the habituation to diffuse photic stimuli was demonstrated to be almost complete in pigeons. Birds submitted to visual Wulst ablation or supraoptic commissure lesion performed in much the same way. In contrast, when the tectal and posterior commissures were lesioned, pigeons failed to show interocular transfer of that learning process.