Neurogenesis of cholinoceptive neurons in the chick retina

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 95 (1996) 205-212

Research report

Neurogenesis of cholinoceptive neurons in the chick retina P.F.

Gardino

~.. C a l a z a a , D . E . H a m a s s a k i - B r i t t o a , K . i~

b J.M. Lindstrom c L.R.G. Britto d

J.N. Hoko~ a,* a Department ofNeurobiology, In.¢titute of Biophysics Carlos Chagas Filho, Center of Health Sciences, Block G, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21949-900, Brazil b Department of Histology and Embryology, Institute of Biomedical Sciences, University ofS~o Paulo, S~o Paulo, SP 05508-900, Brazil c Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104-6074, USA d Department of Physiology and Biophysics, Institute of Biomedical Sciences, University ofSgto Paulo, Sg~oPaulo, SP 05508-900, Brazil

Accepted 2 April 1996

Abstract

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 a3 or the a8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the c~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 c~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 et 8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the a 3-positive cells in the same layer. Keywords: Nicotinic receptor; Alpha-3 subunit; Alpha-8 subunit; Autoradiography; Immunocytochemistry; Cell generation; [3H]Thymidine

1. Introduction

The vertebrate retina contains a prominent cholinergic system, which includes two or three populations of cholinergic amacrine cells [8,11,15,27-30,40,42,43]. Acetylcholine released by those neurons in the inner plexiform layer appears to modulate, among other processes, the directional selectivity of ganglion cells, the activity of enkephalinergic neurons, and the outgrowth of ganglion cell neurites. Such actions; are known to be mediated by nicotinic receptors [1-3,26]. The retinal neurons containing nicotinic receptors have only recently begun to be characterized in detail, after the cloning and sequencing of several subunits that constitute neuronal nicotinic acetylcholine receptors (nAChPs). Both cDNAs and subunitspecific antibodies have become available that permitted studies of several o~ (or ligand-binding) subunits and [3 (non-c~, or structural) subunits of the nAChRs [7,10,25,33,34]. Expression of nAChR genes has been studied in the rat [20,44] and goldfish [6] retinae. Antibod* Corresponding author. Fax: (155) (21) 280-8193.

ies have been used to characterize the cell types that contain nAChRs in the frog [35], rat [41], chick [4,16,17,23,24,45], and squirrel [5] retinae. Both amacrine cells and ganglion cells appear to contain nAChR subunits. Several oL subunits (~3, o~5, oL7, and a 8 ) and at least two [3 subunits ([32 and [34) have been localized to amacrine and ganglion cells, with varying degrees of co-localization [4]. Displaced ganglion cells appear to contain a 3 , e~5, [32, and [34 nAChR subunits [5,17,24]. The diversity of nAChR subunits in retinal neurons suggests that a wide variety of nAChRs are to be found in such neurons, as part of a complex cholinoceptive system in the vertebrate retina. One of the interesting aspects of the diversity of nAChR subunits in the retina is related to their ontogenetic development. Some nAChR genes are known to be developmentally regulated [20]. We have recently demonstrated by immunocytochemistry that nAChR subunits of the abungarotoxin-sensitive type develop at a faster rate than subunits of the a-bungarotoxin-insensitive type in the chick retina [18]. The latter type, however, appears in retinal somata at least one day earlier than the former type of nAChR subunit. Interestingly, both types of nAChR sub-

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units are present in retinal neurons about two days before the onset of acetylcholine synthesis and several days before synaptogenesis, which suggests a role for those receptors in developmental regulation. This conclusion has prompted us to analyze the neurogenesis of the cholinoceptive cells in the retina, in order to verify if the times of birth of these cells differ from the times of birth of the other, non-cholinoceptive cells in their respective classes. As in the previous study, we investigated the two major nAChR e~ subunits in the chick retina, which are the e~3 (of the 'classical', a-bungarotoxin-insensitive type) [45] and the o~8 nAChR subunits (of the a-bungarotoxin-sensitive type) [23]. The characterization of the time of birth of retinal cells containing c~3-1ike and a8-1ike immunoreactivity ( a 3-LI and eL8-LI) has been performed by means of [3H]thymidine autoradiography [13,37].

2. Materials and methods

2.1. Thymidine injection Fertilized White Leghorn eggs were purchased from a local hatchery in Rio de Janeiro. A total of 26 embryos from E1 to E l i received a single injection of 25 ~1 of 1 m C i / m l [3H]thymidine (Amersham, specific activity 46 C i / m m o l ) through a little window made in the shell overlying the embryo. Each retina used in this study was obtained from a different animal. The eggs were sealed with tape, returned to the incubator (37°C, 80% humidity) and resumed development until 17-19 days of incubation (E17-E19), at which time the retina has attained its morphological maturity. The embryos were killed by decapitation and then staged according to Hamburger and Hamilton [19]. The eyeballs were rapidly enucleated, hemisected with a razor blade, and the posterior eyecup with the retina was immersed in fixative (2% paraformaldehyde in 0.16 M phosphate buffer (PB) pH 7.2) for 3 h. After several rinses in PB, the tissue was cryoprotected in 20-30% sucrose in PB overnight. The retina was then dissected out from the sclera and radial sections (10-12 txm thick) from the central region of the eye were mounted on gelatin-coated slides. The retinas were staged according to Coulombre [9]. Sections were collected on slides and stored at - 2 0 ° C until they were processed for immunohistochemistry.

2.2. Immunohistochemistry technique The tissue was processed either with a rat monoclonal antibody against the a 3 nAChR subunit (mAb315) [45] or

a rat monoclonal antibody against the a 8 nAChR subunit (mAb305) [36]. lmmunohistochemistry was conducted according to the avidin-biotin-peroxidase protocols. Radial sections were washed in sodium phosphate-buffered saline (PBS) for 5 min, and incubated with the primary antibody diluted 1:250 in PBS with 0.3% Triton X-100 for 12-24 h at 4°C. After several rinses in PBS, the sections were incubated with a goat anti-rat IgG biotinylated antiserum (Jackson Labs., West Grove, PA) diluted 1:200 in PBS containing 0.3% Triton X-100 for 1 h at room temperature. Further washes in PBS were made and the sections were incubated in a mixture of biotin and avidin-peroxidase (ABC Elite kit, Vector Labs., Burlingame, CA) diluted 1:100 in PBS with 0.3% Triton X-100 for 1 h at room temperature. The sections were rinsed in PBS for 30 min, incubated in 0.05% 3,3'-diaminobenzidine in PBS for 5 min, and a 0.3% solution of hydrogen peroxide in distilled water was then added to reach a final concentration of 0.01%. The reaction proceeded for 10 min, and the sections were finally rinsed in PBS. The slides were allowed to dry and autoradiography was done. Control procedures were the omission of the primary antibodies and substitution of the primary antibodies with rat normal sera. No immunoreactivity was detected in control sections. It should be stressed that the specificity of the antibodies used in this study has been extensively characterized [36,45], and they have previously been used in similar experiments [16,17,23].

2.3. Autoradiography To determine the time of birth of retinal cells, we employed the autoradiographic labeling method, via the incorporation of [3H]thymidine [12,37]. The cumulative labeling method described by Fujita and Horii [13] was used. Briefly, the slides were dipped in Kodak NTB2 emulsion, placed in air-tight, light-tight boxes, and allowed to expose for 2 - 4 weeks at 4°C. The slides were developed in Kodak Dektol developer, fixed, washed, dehydrated, cleared, and coverslipped with Entellan.

2.4. Data analysis Radial sections from E17-E19 retinas were examined under a light microscope (Axioskop, Zeiss). Autoradiographic labeling was investigated taking into account the distinct sizes of retinal cells and the distinct backgrounds. The cell soma areas were calculated by measuring the mean between the longer axis and its perpendicular axis. The background was determined, in each section, by

Fig. I. Photomicrographs of transverse sections of the chick retina illustrating the criteria used to characterize the doubly labeled retinal neurons. The hollow arrows indicate doubly labeled cells, whereas the arrowheads indicate neurons that were only immunocytochemically stained. Many cells are apparent that contained only autoradiographic grains. Thymidine injections were performed at E6 for the retina illustrated in the upper photograph, and at E9 for the retina depicted in the lower photograph. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bar: 30 txm.

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counting silver grains in sample areas equivalent to cell soma areas and placed on a known unlabeled region (in the inner plexiform layer). We used the cumulative labeling method adopted by Fujita and Horii [13]. As the [3H]thymidine injected in ovo remains available to be incorporated into the DNA for several days [37], the presence of unlabeled cells can be interpreted as evidence of a withdrawal from the mitotic cycle prior to thymidine administration. On the other hand, the observation of labeled cells, with grain overlying the nucleus, is usually interpreted as indicative that those cells were generated at the approximate time of isotope administration, with few 'dilutions' through subsequent division [121. In summary, in this study, the cells were considered 'unlabeled' when the number of grains observed over the cell was equivalent to the number in the background. Neurons were only considered 'labeled' when a grain density was observed over the cell that was 3 times or more above that of the background or when the cell soma exhibited a solid mass of grains too dense to be counted as individual grains. Alternate sections were chosen for analysis. The et 3-LI and c~8-LI cells with no autoradiographic grains were assumed to have been generated prior to the time of thymidine injection. Precursors of doubly labeled cells (c~ 3-LI or o~8-LI and silver grains) were assumed to be undergoing mitotic division at the time of the injection.

3. Results The retinas used in this study were obtained from embryos between El7 and El9. There were three reasons for this choice. First, the chick retina attains a morphologically mature pattern with all cell types and layers differentiated and completed around E l 4 [13,21,22]. Second, naturally occurring cell death in the chick retina takes place from E11 to El6 [38]. Third, the number of cells expressing both or3 and oL8 nAChR subunits increases in the chick retina from El2 to El7 [18], and then remains approximately constant to the adult stage. The first step of our analysis was to evaluate the immunocytochemical labeling for nAChR subunits. Alpha3-like immunoreactivity was detected in amacrine, displaced ganglion cells, and cells of the ganglion cell layer, whereas c~8-1ike immunoreactivity was noticed in amacrine, bipolar cells, and cells of the ganglion cell layer. The time of appearance of immunoreactivity confirmed previous results of our group [18]. Retinal sections from embryos injected with [3H]thymidine from E1 through E11 and immunolabeled for a 3 and o~8 nAChR subunits were then processed for autoradiography. Immunolabeled cell bodies were searched for the presence of autoradiographic grains. Cell bodies that contained the o~3 and a 8 subunits and with grains above the background (see Section 2.4) were considered as being generated after the time of

Fig, 2. Photomicrographs of transverse sections of the chick retina, illustrating doubly labeled cells in the inner nuclear layer. The cell indicated by the arrow in the photograph at the top is probably a displaced ganglion cell, whereas the cell indicated in the photograph at the bottom is probably a bipolar cell. Times of thymidine injection were E7 (~3) and E9 (~8). Scale bars: 20 tzm.

[3H]thymidine injection. These neurons will hereafter be referred to as doubly labeled. Cell bodies with no grains or a number of grains below the background were probably generated prior to the time of injection. Figs. 1 and 2 depict photomicrographs of retinal sections that were processed for o~3 and c~8 nAChR immunoreactivity and [3H]thymidine autoradiography, to illustrate the criteria used to characterize doubly and singly labeled cells. Alpha-3-1ike immunoreactive cell bodies in the ganglion cell layer were first detected at E4.5. The soma sizes of these neurons indicated that they comprise two different populations, with the largest axes averaging 8.7 + 1.1 p~m and 13.8 _+ 1.6 ~m. Both types of cells were generated from E2 to E7, according to the autoradiographic label. In the inner nuclear layer, two populations of o~3-positive cells were found, which appeared to correspond to small to medium-sized amacrine cells (8.7 + 1.1 p.m) and medium to large-sized displaced ganglion cells (14.8 _+ 1.7 ~m). The identification of the latter cells as displaced ganglion cells was based on their morphological characteristics [32]

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and on the fact that these cells are known to contain et 3 immunoreactivity in the chick retina [17]. The amacrine cells appeared to have been generated from E2 to E9, whereas the displaced ganglion cells were born from E3 to E9. The time course of generation of both populations was therefore quite similar, although the rates of generation of both populations were different (Fig. 3). For instance, at E5, about 20% of amacrine cells and 60% of displaced ganglion cells were already born, suggesting that the majority of displaced ganglion cells leave the cell cycle earlier than amacrine cells. The generation of a 3-positive cell bodies in the ganglion cell layer ends earlier (E7) than the generation of oL3-positive somata in the inner nuclear layer (E9). The immunocytochemist~ry for the tx 8 nAChR subunit revealed five populations of labeled cell bodies in the chick retina. Two of these populations were found in the ganglion cell layer, and exhibited medium to large (13.4 + 1.6 txm) or small to medium (8.1 _+ 1.1 txm) somata. Three populations of tx 8-LI somata were seen in the inner nuclear layer. Bipolar cells that were tx8-positive were always small (5.7 + 1.0 Ixm), whereas the amacrine cells were either small to medium (8.7 + 1.1 p~m) or medium to large (12.4+ 1.1 txm). Cells containing a 8 - L I in the ganglion cell layer appeared to be generated from E1 through E7. However, only one cell in that layer was observed to be born on E l. This indicates that the vast majority of c~8-positive neurons in the ganglion cell layer leave the cell cycle between E2 and E7. Both populations of tx8-positive amacrine cells were generated from E2 through E9, whereas the time of birth of ix S-positive bipolar cells ranged from E5 to E11 (Fig. 4). As observed for oL3-like immunoreactivlity, the generation of a 8-posifive cells in the ganglion cell layer appeared to be completed (E7) a few days before the generation of ct8-positive cells in the inner nuclear layer ends (E9-E11).

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The neurogenesis of both o~3-LI and oL8-LI neurons in the chick retina followed similar time courses for the specific cell classes, except for one major aspect. The et 8-LI neurons of the ganglion cell layer appeared to start leaving the cell cycle earlier than the tx 3-LI cells in the same layer. Besides the one cell found to leave the cell cycle on El, about 35% of the a8-positive neurons in that layer appeared to have already been born by E4, whereas at this stage less than 10% of the ct3-positive cells in the ganglion cell layer have been born. From E4 through E5, however, the rate of tx3-positive cells leaving the cell cycle is much faster than that of the e~8-positive neurons (Figs. 3 and 4). Interestingly, both types of tx 8-containing amacrine cells also appear to leave the cell cycle at a somewhat faster rate than the oL3-positive amacrine cells, although the range of their birthdates is similar.

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The pattern of a 3 - L I and et8-LI found in this study with retinae from El7 through El9 was essentially similar to the pattern described for the adult chick retina [16,17,23]. The autoradiographic results of this study indicate that the neurogenesis of cells exhibiting o~3-LI and c~8-LI follows the general pattern of cell generation in the chick retina [13,22,31,32,39]. The periods of generation of immunolabeled cells in the ganglion cell layer (E2-E7), amacrine cells (E2-E9), and bipolar cells (E5-E11) are all in agreement with those previous neurogenetic data. It is worth mentioning that there is partial co-localization of ~3 and ~x8 nAChR subunits in the chick retina. Indeed, despite the fact that these two subunits do not appear to form receptor complexes together [36], they have been co-localized in a few cases to the same cells [4]. This indicates that a few

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cells that were included in the a 3 - or the a8-positive populations could actually be the same neurons. At least one difference was noticed between the neurogenesis of c~3- and e~8-positive neurons in the chick retina. The cells in the ganglion cell layer that stained for a 3 - L I begin to leave the cell cycle almost one day later than the a8-positive neurons. Furthermore, the e~3-positive cells were observed to leave the cell cycle at a slower rate than the ~ 8-positive cells, until E4. Yet the expression of the c~3 protein in the chick retina appears to start one day before the expression of c~8 [18]. These results suggest that the factors controlling expression of c~3 and c~8 nAChR subunits in the chick retina appear to be relatively independent of those that control cell genesis. Indeed, the ontogenesis of c~3 and e~8 nAChR subunits in the chick retina is not related in any simple way to the neurogenesis of neurons bearing those receptor subunits. As depicted in Fig. 5, ~8-LI in bipolar cells and a 3 - L I in displaced ganglion cells are both detectable only after the neurogenesis of those cells is completed, whereas both a 3-LI and o~8-LI in amacrine cells and cells of the ganglion cell layer are detectable 2 - 3 days before the end of mitosis of those neurons. The rates of neurogenesis of e~3- and o~8-positive

amacrine cells also appear to differ slightly, although in this case no difference in their ontogenesis has been noted in a previous study [18]. The ~x3-positive presumptive displaced ganglion cells were found in this study to leave the cell cycle between E3 through E9. This period is similar to the period of neurogenesis already determined for displaced ganglion cells in the chick retina [32]. It should be stressed that there could be a problem with the identification of the displaced ganglion cells. In fact, the criteria used to characterize those cells have been mainly based on the soma size and a few other morphological characteristics of displaced ganglion cells [17,32]. In our material, some of the largest a3-positive amacrine cells could be misidentified as a3-positive displaced ganglion cells, and the smaller of the latter cells might have been misidentified as amacrine cells. Retrograde tracing appears to be the only accurate method to identify displaced ganglion cells, but the use of such techniques in developmental studies poses several difficulties. One possibility could be the use of carbocyanine dyes applied to the optic nerve [38]. A similar problem of identification exists in relation to the displaced amacrine cells. Indeed, there is no informa-

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P.F. Gardino et al. / Developmental Brain Research 95 (1996) 205-212

tion on the possibility that at least part of the ct 3- and cx 8-containing cells in the g a n g l i o n cell layer of the chick retina could be displaced anmcrine cells [16,17,23]. These cells appear to originate b e t w e e n E5.5 and E6.5, w h e n identified by cresyl violet and Golgi staining [14,39]. This period of neurogenesis overlaps extensively with the second half of the period of neurogenesis of et 3 and cx 8-positive neurons in the ganglion cell layer. Interestingly, most of the displaced amacrine cells in the chick retina appear to be cholinergic [40], and. no co-localization has b e e n detected b e t w e e n the cholinergic and the cholinoceptive phenotypes in the chick retina [24]. This indicates that very few displaced amacrine cells should have n A C h R s in that species, and most o f the i m m u n o h i s t o c h e m i c a l l y stained cells in the ganglion cell layer might then be g a n g l i o n cells. This point clearly deserves further investigation, with the aid of retrograde tracing and optic nerve lesion techniques. The present results c o n f i ; m that the cholinoceptive system of the chick retina develops very early. The data also reveal that there are some differences in the times of birth of cx3- and cx8-positive cholinoceptive neurons, despite the fact that the neurogenesis of both cell types follows the general pattern o f cell generation in the chick retina.

Acknowledgements This study was supported b y C N P q (J.N.H., P.F.G., D.E.H.B., L.R.G.B.), F A P E S P (L.R.G.B. and D.E.H.B.), C E P E G (J.N.H. and P.F.G.), F I N E P (J.N.H., D.E.H.B,, L.R.G.B.), N I N D S N S 1 1 3 2 3 (J.M.L.), C o u n c i l for Smokeless T o b a c c o Research (J.M.L.), and M u s c u l a r Dystrophy Association (J.M.L.). The authors also thank A d i l s o n S. Alves and Mair M.M. de C)liveira for technical assistance. K.C.C. was the recipient of a fellowship from CNPq.

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