Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells.

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 microM) was essentially without effect at DIV4 and DIV5, while dihydro-ss-erythroidine (10-100 microM) blocked the response to acetylcholine (3.0 nM-2.0 microM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Ionotropic glutamate receptors during the development of the chick retina.

Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.

Calretinin co-localizes with the NMDA receptor subunit NR1 in cholinergic amacrine cells of the rat retina.

Immunohistochemistry was used to verify whether choline-acetiltransferase colocalizes with calcium-binding proteins and NMDA receptor subunit NR1 in the rat retina. Whereas calbindin and parvalbumin were not observed in cholinergic amacrine cells, calretinin and NR1 were very frequently colocalized with ChAT. Calretinin/NR1-positive cells were also shown, suggesting that calretinin in cholinergic cells of the rat may be related to the buffering of excess intracellular calcium generated by activation of NMDA receptors.

A comparative non-radioactive in situ hybridization and immunohistochemical study of the distribution of alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors in visual areas of the chick brain.

The distribution of mRNA transcripts corresponding to the alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors (nAChRs) was studied in selected structures of the chick visual system with non-radioactive in situ hybridization and immunohistochemical techniques. The results indicated that the alpha7 and alpha8 nAChR transcripts are widely distributed in the brain, exhibiting differential expression in some structures but also some degree of co-localization. The pattern of localization of alpha7 and alpha8 nAChR transcripts was highly correlated with immunohistochemical data, with very few instances of possible mismatches between the distribution of mRNAs and their corresponding proteins.

Motion-sensitive neurons in the chick retina: a study using Fos immunohistochemistry.

Fos immunohistochemistry was used to characterize neurons in the chick retina activated by optokinetic and stationary stimuli. Higher percentages of co-localization of Fos and the alpha5 subunit of the nicotinic acetylcholine receptor, and Fos and GABA were observed in retinal neurons after optokinetic compared to the stationary stimulation. These results indicate an involvement of the cholinergic and GABAergic circuitries in the motion detection by chick retinal cells.

Differential co-localization of nicotinic acetylcholine receptor subunits with calcium-binding proteins in retinal ganglion cells.

Immunohistochemistry was used to examine the co-occurrence of nicotinic acetylcholine receptor subunits with calcium-binding proteins in ganglion cells of the chick retina. The alpha3 subunit was rarely observed in ganglion cells containing calbindin, calretinin, or parvalbumin. On the other hand, the alpha8 subunit was more often co-localized with all calcium-binding proteins studied. These results may be related to the high calcium permeability of nicotinic receptors that contain the alpha8 subunit.

Expression of the Fos protein reveals functional subdivisions of the avian ventral lateral geniculate nucleus.

The Fos protein was immunocytochemically detected in the chick ventral lateral geniculate nucleus after novel stationary and optokinetic stimulation. Fos-positive nuclei were mainly detected in the internal part of the ventral geniculate when the animals were submitted to stationary visual stimulation. On the other hand, Fos-positive nuclei were mainly seen in the external part of the nucleus when optokinetic stimuli were used. These data reveal functional subdivisions of the avian ventral geniculate, and support the hypothesis that this nucleus is involved in several aspects of the visual function.

Neurogenesis of cholinoceptive neurons in the chick retina.

Immunocytochemistry and [3H]thymidine autoradiography were combined in this study to determine the neurogenesis of cholinoceptive cells in the chick retina. After injections of [3H]thymidine between embryonic days 1 and 11, the time of birth of retinal neurons containing either the alpha 3 or the alpha 8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the alpha 3-positive neurons in the ganglion cell layer leave the cell cycle from E2 through E7, and those in the inner nuclear layer (amacrine and displaced ganglion cells) from E2 through E9. The alpha 8-positive cells in the ganglion cell layer were born from E1 through E7, and those in the inner nuclear layer (amacrine and bipolar cells) from E2 through E11. These data suggest that the time of birth of cholinoceptive neurons in the chick retina follows the general pattern of cell generation in the chick retina, and that alpha 8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the alpha 3-positive cells in the same layer.

Serotonergic afferents of the pigeon accessory optic nucleus.

Injections of a retrograde tracer into the accessory optic nucleus of the basal optic root (nBOR) of the pigeon, combined with 5-HT immunochemistry, revealed that serotonergic projections to the nBOR appeared to originate mainly from the median (MR) and paramedian (PMR) raphe nuclei. These projections were confirmed by the significant decrease in 5-HT immunoreactivity observed in nBOR after lesions in MR and PMR. These data characterize distinct sources of 5-HT innervation to the pigeon nBOR and suggest that those afferents could represent part of a modulatory system that contributes to the role of the nBOR in optokinetic mechanisms.

Changes in expression of glutamate receptor subunits following photoreceptor degeneration in the rd mouse retina.

The effects of photoreceptor cell degeneration on the expression of nine alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/kainate glutamate receptor subunit genes were investigated in the rd mouse retina by in situ hybridization. All known AMPA/kainate receptor subunits were found to be expressed in normal mouse retina. Following retinal degeneration, the expression of KA-1 was reduced, that of the GluR7 subunit was greatly increased, and that of the other seven subunits were not significantly affected.

Effects of retinal lesions upon the distribution of nicotinic acetylcholine receptor subunits in the chick visual system.

Immunohistochemistry was used in this study to evaluate the effects of retinal lesions upon the distribution of neuronal nicotinic acetylcholine receptor subunits in the chick visual system. Following unilateral retinal lesions, the neuropil staining with an antibody against the beta 2 receptor subunit, a major component of alpha-bungarotoxin-insensitive nicotinic receptors, was dramatically reduced or completely eliminated in all of the contralateral retinorecipient structures. On the other hand, neuropil staining with antibodies against two alpha-bungarotoxin-sensitive receptor subunits, alpha 7 and alpha 8, was only slightly affected after retinal lesions. Decreased neuropil staining for alpha 7-like immunoreactivity was only observed in the nucleus of the basal optic root and layers 2-4 and 7 of the optic tectum. For alpha 8-like immunoreactivity, slight reduction of neuropil staining could be observed in the ventral geniculate complex, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, and nucleus suprachiasmaticus. Taken together with previous data on the localization of nicotinic receptors in the retina, the present results indicate that the beta 2 subunit is transported from retinal ganglion cells to their central targets, whereas the alpha 7 and alpha 8 subunit immunoreactivity appears to have a central origin. The source of these immunoreactivities could be, at least in part, the stained perikarya that were observed to contain alpha 7 and alpha 8 subunits in all retinorecipient areas. In agreement with this hypothesis, the beta 2 subunit of the nicotinic acetylcholine receptors was not frequently found in perikarya of the same areas.

Differential development of alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina.

The development of cells containing neuronal nicotinic receptors (nAChRs) in the chick retina was investigated by means of immunohistochemical techniques with antibodies directed against the alpha 3 and alpha 8 nAChR subunits. The alpha 3 subunit is one of the major alpha-bungarotoxin-insensitive nicotinic receptor subunits in the chick retina, whereas alpha 8 appears to be the most common alpha-bungarotoxin-sensitive subunit in the same structure, alpha 3-like immunoreactivity (alpha 3-LI) was first detected in cells of the vitreal margin, on the embryonic day 4.5 (E4.5). alpha 8-LI was first detected in the same type of cell almost a day later. However, the processes of alpha 8-LI cells developed much faster than those of alpha 3-LI cells, generating visible stained laminae in the prospective inner plexiform layer as early as E7. alpha 3-LI was only clearly seen in laminae of the inner plexiform layer by E12. By this date, both alpha 3 and alpha 8-LI were seen in the same types of cells as in the adult retina, i.e., amacrines, displaced ganglion cells, and cells of the ganglion cell layer for alpha 3-LI; and amacrines, bipolar cells, and cells of the ganglion cell layer for alpha 8-LI. These results reveal different patterns of development of cells containing the alpha 3 and alpha 8 nAChR subunits in the chick retina and indicate that those nAChR subunits are expressed in the chick retina before choline acetyltransferase-positive cells can be detected and well before synaptogenesis. These data also suggest that nAChRs may have a developmental function in the retina.

Nicotinic acetylcholine receptors in the ground squirrel retina: localization of the beta 4 subunit by immunohistochemistry and in situ hybridization.

Immunohistochemical and in situ hybridization techniques were used to localize the beta 4 subunit of the neuronal nicotinic acetylcholine receptors (nAChRs) in the ground squirrel retina. The beta 4 nAChR subunit was detected in both transverse and horizontal sections of the retina using a subunit-specific antiserum and the avidin-biotin complex technique. Two bands of labeled processes were seen in the inner plexiform layer, corresponding approximately to the laminae where the cholinergic cells arborize. Labeled cells were found in the ganglion cell layer and the inner third of the inner nuclear layer. The cells in the ganglion cell layer were medium- to large-sized and were frequently observed to give rise to axon-like processes. Most of the labeled neurons in the inner nuclear layer were small presumptive amacrine cells, but a few medium-to-large cells were also labeled. These could constitute a different class of amacrine cells or displaced ganglion cells. The latter possibility is supported by the existence of nAChR-containing displaced ganglion cells in the avian retina. In situ hybridization with a 35S-labeled cRNA probe revealed the expression of mRNA coding for the nAChR beta 4 subunit in the ganglion cell layer and the inner third of the inner nuclear layer. This finding confirmed the immunohistochemical data of the cellular localization of beta 4 nAChR subunit. These results indicate that the beta 4 nAChR subunit is expressed by specific subtypes of neurons on the ground squirrel retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Bipolar cells of the chick retina containing alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors.

Two cDNA clones for nicotinic acetylcholine receptor (nAChR) subunits sensitive to alpha-bungarotoxin (alpha-Bgt) have been isolated, the so-called alpha-Bgt binding proteins alpha 1 (or alpha 7 nAChR subunit) and alpha 2 (or alpha 8 nAChR subunit). Immunohistochemical experiments have shown that both alpha 7 and alpha 8 subunits, as well as subunits insensitive to alpha-Bgt (beta 2 and alpha 3), are present in amacrine and ganglion cells of the chick retina. However, only the alpha 8 subunit was observed in presumptive bipolar cells. The present study investigated in detail the pattern of distribution of the bipolar cells containing the alpha 8 nAChR subunit and its relation to the pattern of distribution of bipolar cells immunoreactive to protein kinase C (PKC). Presumptive alpha 8- and PKC-like immunoreactive (alpha 8-LI and PKC-LI) bipolar cells were observed sending their dendrites to the outer plexiform layers and their axons to the inner plexiform layer. Whereas alpha 8-LI bipolar cells corresponded to 40-53% of the whole population of bipolar cells, PKC-LI bipolar cells represented only 6-8% of the same population. The soma sizes of the alpha 8-LI bipolar cells were slightly smaller (mean +/- S.D.; 4.9 +/- 0.8 microns) than the soma sizes of the PKC-LI bipolar cells (5.4 +/- 0.9 microns). Double-labeling experiments indicated that probably all PKC-LI bipolar cells also contain alpha 8-LI. This indicates that two distinct groups of cholinoceptive bipolar cells exist in the chick retina, one that contains PKC-LI, and another one that does not.

Expression of glutamate receptor genes in the mammalian retina: the localization of GluR1 through GluR7 mRNAs.

Seven distinct cDNAs encoding functional subunits of the AMPA/kainate-type glutamate receptors have been recently cloned. This in situ hybridization study was done to determine which subunits are expressed in the retina and, where possible, which neurons express them. Hybridization of 35S-UTP-labeled cRNA probes with transverse sections revealed that mRNAs for all seven receptor subunits (GluR1-GluR7) are expressed in both cat and rat retinas. GluR1 and GluR2 produced labeling over the entire inner nuclear layer (INL) and ganglion cell layer (GCL). GluR3-GluR7 have more limited distributions, indicative of expression by only a subset of neurons. All of the subunits are expressed by the cells at the inner edge of the INL, where amacrine cells reside, yet the layers with the horizontal, bipolar, and ganglion cells contain different subsets of subunits. These findings suggest that these glutamate receptor subunits are employed at many of the retinal synapses, including the photoreceptor input to the outer plexiform layer and the bipolar cell's contacts with the processes at the INL. It is also possible that some glial cells in the INL express some of the subunits. Since different combinations of GluR1-GluR3 have been shown to play an important role in the calcium permeability in response to glutamate, we investigated whether single cells coexpressed those subunits. By hybridizing adjacent semithin (1 micron) sections of the cat retina with probes for GluR1-GluR3, it was possible to observe coexpression of all three subunits, or of pairs of these subunits, in cells within the INL and GCL.

Different subsets of displaced ganglion cells in the pigeon retina exhibit cholecystokinin-like and enkephalin-like immunoreactivities.

Immunohistochemical and retrograde tracing techniques were combined in order to identify chemically specific displaced ganglion cells in the pigeon retina. About 15% of the displaced ganglion cells that were retrogradely labeled following injections of different tracers into the accessory optic nucleus were shown to contain cholecystokinin8-like immunoreactivity. These cells were medium to large (15-30 microns) and located mostly in the peripheral retina. Another population of about 9% of the retrogradely labeled displaced ganglion cells was shown to contain leucine-enkephalin-like immunoreactivity. These cells were medium-sized (11-18 microns) and distributed almost evenly throughout the retina. These two types of displaced ganglion cells represent together only about 0.1% of the total number of ganglion cells in the pigeon retina. Taken together with previous results, these data indicate that the displaced ganglion cells of the avian retina may comprise several chemically specific cell types. The present results also contribute information on the chemical heterogeneity of retinal ganglion cells.

Neurons of the chick brain and retina expressing both alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis.

Immunohistochemical methods were used to study the possible co-localization of two alpha-bungarotoxin-sensitive (alpha 7 and alpha 8) and two alpha-bungarotoxin-insensitive (beta 2 and alpha 3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the alpha 7 subunit and the beta 2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the alpha 7 and alpha 8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain alpha 8 and alpha 3 subunits, whereas neurons in the ganglion cell layer were seen to contain alpha 7 and alpha 8 or, less frequently, alpha 7 and alpha 3 subunits. These results indicate that alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.

Enkephalin-immunoreactive ganglion cells in the pigeon retina.

A small number of enkephalin-like immunoreactive cells were observed in the ganglion cell layer of the pigeon retina. Many of these neurons were identified as ganglion cells, since they were retrogradely labeled after injections of fluorescent latex microspheres in the contralateral optic tectum. These ganglion cells were mainly distributed in the inferior retina, and their soma sizes ranged from 12-26 microns in the largest axis. The enkephalin-containing ganglion cells appear to represent only a very small percentage of the ganglion cells projecting to the optic tectum (less than 0.1%). Two to 7 weeks after removal of the neural retina, there was an almost complete elimination of an enkephalin-like immunoreactive plexus in layer 3 of the contralateral, rostrodorsal optic tectum. These data provide evidence for the existence of a population of enkephalinergic retinal ganglion cells with projections to the optic tectum.

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 microns. Some double-labeled cells ranging from 7-21 microns were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells.