Dânia E. Hamassaki-Britto

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

Abstract Immunohistochemical methods were used to study the possible co-localization of two α-bungarotoxin-sensitive (α7 and α8) and two α-bungarotoxin-insensitive (β2 and α3) 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 α7 subunit and the β2 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 α7 and α8 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 α8 and α3 subunits, whereas neurons in the ganglion cell layer were seen to contain α7 and α8 or, less frequently, α7 and α3 subunits. These results indicate that α-bungarotoxin-sensitive and α-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.

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.

Bipolar cells of the chick retina containing α-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.

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 (α‐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.J. Comp. Neurol. 441:58–70, 2001.

Nicotinic acetylcholine receptors in the ground squirrel retina: localization of the β4 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)

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina.

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.

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.

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.

A comparative non-radioactive in situ hybridization and immunohistochemical study of the distribution of α7 and α8 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.

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.