Cell Tissue Res (2000) 302:11–19 DOI 10.1007/s004410000267
REGULAR ARTICLE
Silke Haverkamp · Helga Kolb · Nicolas Cuenca
Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina
Received: 22 May 2000 / Accepted: 17 July 2000 / Published online: 24 August 2000 © Springer-Verlag 2000
Abstract The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina. This study was supported by grant EY04885 to H. Kolb S. Haverkamp Max-Planck-Institut für Hirnforschung, D-60528 Frankfurt, Germany H. Kolb (✉) John Moran Eye Center, University of Utah Health Sciences Center 75 N., Medical Drive, Salt Lake City, Utah 84032, USA e-mail:
[email protected] N. Cuenca Biotechnology Department, University of Alicante, Alicante, Spain
Key words Neuronal nitric oxide synthase · Reduced nicotinamide adenine dinucleotide phosphate diaphorase · GABA · Glycine · Calbindin · Immunocytochemistry · Pseudemys scripta elegans (Chelonia)
Introduction Nitric oxide (NO) has been shown to function as a messenger molecule in most tissues. In the retina, previous studies have indicated that NO modulates light transduction and signal transmission, e.g., by modifying photoreceptor transmitter release (Kurenni et al. 1995; Savchenko et al. 1997) or affecting responsiveness and coupling of horizontal cells (Miyachi et al. 1990; Pottek et al. 1997; Cudeiro and Rivadulla 1999 for a review). NO also stimulates the soluble isoform of guanylate cyclase, which in turn synthesizes cyclic guanosine monophosphate (cGMP), so that cGMP-gated conductances in On bipolar cells (Shiells and Falk 1992) and ganglion cells (Ahmad et al. 1994) can be modulated by NO. Three isoforms of nitric oxide synthase (NOS), the enzyme responsible for generating NO from L-arginine, have been characterized: the neuronal and endothelial isoforms (nNOS and eNOS) are calcium-dependent, constitutive proteins, which require reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor, while the other one is calcium-independent and inducible (iNOS; Förstermann et al. 1994). NOS can be localized directly by immunocytochemistry or indirectly by NADPH diaphorase (NADPH-d) histochemistry (Dawson et al. 1991; Hope et al. 1991). Several investigations have used these two markers to identify NO-synthesizing neurons in the retinas of various species (Sandell 1985; Wässle et al. 1987; Yamamoto et al. 1993; Weiler and Kewitz 1993; Liepe et al. 1994; Koistinaho and Sagar 1995; Kurenni et al. 1995; Perez et al. 1995; Blute et al. 1997; Haverkamp and Eldred 1998). Amacrine cells are the commonest cell types labeling to nNOS and in some species photoreceptors, horizontal cells, bipolar cells, and ganglion cells are also
12 Table 1 Antisera and technical information (nNOS neuronal nitric oxide synthase, TH tyrosine hydroxylase, ChAT choline acetyltransferase, PV parvalbumin) Antigen
Antiserum
Source
Working dilution
bNOS 1–181 nNOS 1400–1419 (rat) GABA GABA Glycine Tyrosine hydroxylase Choline acetyltransferase Calretinin Calbindin Parvalbumin Somatostatin
Mouse anti-bNOS Rabbit anti-nNOS Rabbit anti-GABA Rat anti-GABA Rat anti-glycine Rabbit anti-TH Rabbit anti-ChAT Rabbit anti-calretinin Rabbit anti-calbindin Rabbit anti-PV Mouse anti-somatostatin
Sigma Immunochemicals, St. Louis, MO, USA Santa Cruz Biotechnology, Santa Cruz, CA, USA D. Pow, Brisbane, Australia D. Pow D. Pow Chemicon International, Temecula, CA, USA Chemicon International, Temecula, CA, USA Swant, Bellinzona, Switzerland Swant, Bellinzona, Switzerland Swant, Bellinzona, Switzerland Pharmingen International, San Diego, CA, USA
1:100 1:500 1:1000 1:1000 1:1000 1:400 1:500 1:2000 1:2000 1:1000 1:100
Materials and methods Four turtles (Pseudemys scripta elegans) were used for this study. All procedures followed the guidelines of the University of Utah IACU for the use of turtles in acquiring histological data. The animals were decapitated and their eyes enucleated and hemisected along the vertical midline. The resultant eyecups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 30–40 min at room temperature. Following fixation, all eyecups were gradually infiltrated with increasing sucrose concentrations before embedding in OCT and cutting into 14-µm-thick radial sections using a cryostat. Sections were collected on gelatinized slides, air dried, and stored at –20°C until they were processed for diaphorase histochemistry and immunostaining. For immunofluorescent double-labeling, the cryostat sections were incubated overnight in a cocktail of two primary antibodies (Table 1; antiserum in PB plus 5% normal goat serum and 0.5% Triton X-100). Following several washes, the sections were then incubated in FITC-conjugated anti-rat or anti-mouse IgG (1:100) and rhodamine-conjugated anti-rabbit IgG (1:100; Jackson Immunoresearch) for 60 min. After washing and coverslipping, slides were viewed using a confocal microscope (Zeiss 510). Pinholes were 77 µm and the widths of optical sections were 0.9 µm. Images were transferred into Adobe Photoshop and printed on an Epson ink jet printer. Controls were run by omitting the primary antibody in the immunostaining procedures. No labeling occurred. For NADPH-d histochemistry, the sections were first incubated in a solution of 1 mg/ml NADPH with 0.1 mg/ml of nitroblue tetrazolium (NBT) in 1% Triton X-100 for 1–2 h at 37°C in dark. Following several washes, the sections were then processed for immunocytochemistry as described above.
Fig. 1a–j Confocal microscopic images of vertical cryostat sections through turtle retinae that were double labeled for neuronal nitric oxide synthase (nNOS; rhodamine; red channel) and either glycine (fluorescein; green channel in a–d) or GABA (fluorescein; green channel in e–j). The double-labeled cells appeared orange in the confocal microscope. The images demonstrate the variety of nNOS-stained amacrine cells that were found. Five morphologically distinct subsets were identified: type-1 glycinergic amacrines (GLY+) in b and c; type-2 GLY+ amacrine cells in a and d; type-4 GABAergic amacrines (GABA+) in e–g; and type5 GABA+ cells in i and j. h An additional GABA+ cell was occasionally seen in the ganglion cell layer (GCL), but it was unclear which type it was or whether it was a ganglion cell instead (compare schematic drawing in Fig. 5 and see text for full description). (OPL Outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, H1 H1 horizontal cell soma, HAT axon terminal of H1). Bar 20 µm
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marked. eNOS is seen in some species in Müller cells and axon terminals of H1 horizontal cells in the turtle retina (Haverkamp et al. 1999). Amacrine cells are third-order interneurons of the vertebrate retina that come in a wide variety of morphologies, especially in their dendritic tree extent and stratification. Based on Golgi studies and intracellular dye injections, 37 different morphological types are found in turtle retina (Kolb 1982; Ammermüller and Kolb 1996). These can be distinguished into small-field, mediumfield, and wide-field varieties, with different stratification levels in the inner plexiform layer (IPL). nNOS immunoreactivity has been localized in three amacrine cell varieties in the turtle retina (Blute et al. 1997), but they have not been identified as to type yet and their other conventional neurotransmitter remains unidentified. In other areas of the central nervous system, NADPHd-stained neurons colocalize a variety of neurotransmitters: e.g., with acetylcholine in the basal forebrain (Pasqualotto and Vincent 1991), with GABA in the cerebral cortex (Valtschanoff et al. 1993), and with serotonin in the dorsal raphe nucleus (Johnson and Ma 1993). Furthermore, GABA immunoreactivity has been shown in NADPH-d-stained horizontal cells in carp retina (Weiler and Kewitz 1993) and in NADPH-d-stained amacrine cells in cat, rabbit, and rat retinas (Müller et al. 1988; Vaney and Young 1988; Oh et al. 1998). In the chick retina, which is more closely related to turtle retina than the above mammalian retinas, Fischer and Stell (1999) describe four morphologically distinct types of nNOS-containing amacrine cells, one immunoreactive for parvalbumin, one for somatostatin, and one for glucagon. The aim of this study was: (1) to get a more precise morphological description of nNOS-/NADPH-d-stained amacrine cells in the turtle retina than has been available before; (2) to investigate whether these cells colocalize the inhibitory transmitters GABA or glycine; and (3) to see whether they colocalize any of the other commonly described amacrine cell markers such as catecholamines and calcium-binding proteins.
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Results nNOS-IR amacrine cells colocalize GABA or glycine
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Double immunofluorescence experiments with antibodies against nNOS and either GABA or glycine showed that nNOS-immunolabeled amacrine cells in turtle retina can either be GABA or glycine-IR. Figure 1 demonstrates the variety of nNOS-labeled amacrine cells that we found in this study. At least five morphologically distinct subtypes were identified. The double-labeling experiments with nNOS and glycine (Fig. 1a–d) revealed at least two glycinergic types. One small-field bushy type had processes leaving the soma and multistratifying in the IPL (Fig. 1b,c) and was named “type 1”. The other one had either a short, single apical dendrite or several dendrites leaving the cell body to run with a looser, slightly wider dendritic tree throughout the IPL (Fig. 1a,d) and was named “type 2”. In double-labeling experiments for nNOS and GABA (Fig. 1e–j), we found at least three GABAergic subtypes. GABA-IR was also expressed in the cell bodies and axon terminals of H1 horizontal cells, in amacrine cells located in the inner nuclear layer (INL), and in a few cell bodies in the ganglion cell layer (GCL). Of the GABAergic/nNOS-IR amacrine cells, one type emitted a single apical dendrite that appeared to be bistratified on the S1/2 and 3/4 borders (“type 3”). Type 3 may be represented by the noncolocalized cell in the glycine-IR preparation of Fig. 1d (red cell on the left). The second GABAergic type emitted two or more processes that ran in wide-spread fashion through the IPL to finish running for long distances in S3 (“type 4”; Fig. 1e–g). Still a third type of GABA/nNOS-IR cell was characterized by a very large cell body that sat in the row of cell bodies above the usual amacrine cell layer (Fig. 1i,j). Its single thick apical dendrite split into secondary dendrites that extend in S2 of the IPL. An interesting observation concerns the staining pattern of the nucleus in this variety (“type 5”), where a ring of immunostain is apparent (Fig. 1i,j). In a few instances, NADPH-d-positive cells were seen in the GCL that colocalized GABA (Fig. 1h). The dendrites of these cells were not strongly labeled, and it was not possible to detect where their dendrites ramify. Nor were axons in the ganglion cell layer evident, so we were not sure whether these were displaced amacrine cells or nNOS-IR ganglion cells.
Fig. 2a–l Confocal microscopic images of four vertical cryostat sections that were triple labeled with reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (left column), antiGABA (middle column), and antiglycine (right column). The diaphorase-stained amacrine cell in a was GABA-IR (arrow in b) and corresponds to type 5; the stained cell in d was also GABA-IR (arrow in e) and corresponds to type 4. The NADPH-d-stained amacrine cells in g and j were both glycine-IR (arrows in i and l) and correspond to type 1 (g) and type 2 (j). Bar a–j 20 µm
NADPH-d histochemistry combined with GABA and glycine immunocytochemistry In order to quantify the number of GABAergic and glycinergic cells colocalizing nNOS, triple-labeling experiments for NADPH-d, GABA, and glycine were performed. About 400 NADPH-d-labeled cells were examined: 48% colocalized GABA and 52% glycine. No cells were both GABA- and glycine-IR. Figure 2 shows examples of NADPH-d-stained amacrine cells tested for GABA- and glycine-IR. With the NADPH-d staining, we found the same variety of amacrine cell types as we saw with the nNOS immunostaining in Fig. 1. The NADPH-d-stained cell in Fig. 2a was GABA-IR (arrow in Fig. 2b) but not glycine-IR (Fig. 2c). It is probably the type-5 amacrine cell with dendrites stratifying in S2. Another cell is similarly GABAergic but not glycinergic in Fig. 2d–f, where only the primary dendrites show up. It resembles most the type-4 cell. Of the glycine-immunolabeled cells shown in Fig. 2, one resembles a type 1 (Fig. 2g–i, arrow) and the other a type 2 (Fig. 2j–l, arrow). Further examples of NADPH-d-stained amacrines and their colocalized neurotransmitter are shown in Fig. 3: type 1 in Fig. 3e; type 2 in Fig. 3g,h; type 3 in Fig. 3d,f; type 4 in Fig. 3a,b; and type 5 in Fig. 3c. Colocalization with calbindin We tested other common amacrine cell markers for colocalization with nNOS-immunostained cells in the turtle retina. Figure 4 shows three examples of nNOS-IR cells (Fig. 4a,c,e, arrows) that colocalized calbindin (Fig. 4b,d,f, arrows). Calbindin-IR was found in photoreceptors, a few bipolar cells, and some brightly fluorescent normal and displaced amacrine cells which stratified most likely in the two strongly stained bands in the IPL (Fig. 4b,d,f). The nNOS-IR cells colocalized in a few cases with some of the weakly immunostained calbindin amacrine cells (arrows in Fig. 4a–f). Altogether 26 of 174 (15%) examined nNOS-labeled cells colocalized in calbindin-positive amacrine cells. Since the morphology of these double-labeled cells was quite similar to some of the GABAergic/NADPH-d-stained amacrine cells, and considering the fact that calbindin-IR amacrine cells in general are GABAergic (95% in our experience), we suggest that these cells probably colocalized with GABA as well. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize the nNOS-IR or NADPH-dstained amacrine cells in our experiments (negative data not shown).
Discussion In this study we have been able to show that at least five different morphological types of amacrine cell in the tur-
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Fig. 4a–f Confocal microscopic images of three vertical sections that were double labeled with antibodies against nNOS (top line) and calbindin (bottom line). Some of the weaker-labeled, calbin-
din-positive amacrine cells were also immunoreactive for nNOS (arrows). Bar 50 µm
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Fig. 5 Schematic drawing of the morphological types of nNOS-IR/NADPH-d-stained amacrine cells that colocalize GABA or glycine. One GABA+ displaced cell in the GCL is not classifiable because of poor dendritic staining. It could be a ganglion cell
Fig. 3a–h Examples of diaphorase-stained amacrine cells that were found to be either GABAergic or glycinergic in the triple-label experiments. The cells in a and b belong to type 4, the cell in c is the type 5, and d and f are type 3. The cell in e is a type 1, and the two in g and h are type 2. Bar 20 µm
tle retina are nNOS /NADPH-d-IR and use either GABA or glycine inhibitory neurotransmitters. Here we show that GABA labeled three morphologically different types of NADPH-d amacrine and glycine labeled two types. The five types are schematized in Fig. 5. In comparison
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with amacrine cells revealed in a Golgi study on turtle retina (Kolb 1982), we consider type-1 cells to be A1 cells, type 2 to be A6 cells, type 3 to be A15, type 4 to be A20, and type 5 to be equivalent to A22 cells of turtle retina. GABAergic/NADPH-d-labeled amacrine cells have been shown in cat, rabbit, and rat retina (Müller et al. 1988; Vaney and Young 1988; Oh et al. 1998) but not in turtle heretofore, and glycine colocalizing with nNOS has not been reported in any vertebrate retina to date. Here we show that about half of the NADPH-d-labeled amacrine cells colocalize in glycinergic amacrine cells. Our data extend previous reports on NOS expression in the turtle retina (Blute et al. 1997; Haverkamp and Eldred 1998). Blute and coworkers (1997) distinguished three types of NADPH-d-positive cells in the turtle retina (ND1, ND2, ND3). By comparing the morphology of these authors’ cells with ours in Fig. 5, it seems very likely that the large ND3 resembles our type-4 amacrine cell. The ND2 amacrine cells with smaller, lightly stained somata and more diffusely branching dendrites are similar to our glycinergic type-1 amacrine cells, and the ND1 cells with dendrites in S1, S3, and S4–5 might resemble our type-2 amacrine cells (Blute et al. 1997). Different effects of NO in neurons are suspected: for example NO has been shown to induce neurotransmitter release from hippocampal slices (Lonart et al. 1992) and to modulate endogenous dopamine release in bovine retina (Bugnon et al. 1994). NO has a well known effect in modulating gap junctions between horizontal cells and amacrine cells in the retina through an effect on cGMP pathways (Miyachi et al. 1990; Lu and McMahon 1997; Pottek et al. 1997; Baldridge et al. 1998). Furthermore, a direct effect on the glutamate receptors on horizontal cells has been demonstrated (McMahon and Schmidt 1999). It has been shown that NO can evoke GABA release (McMahon and Ponomareva 1996; Ohkuma et al. 1996; Wexler et al. 1998) and affect cholinergic amacrine activity (Neal et al. 1997), so we suggest that it plays a role in modulating the other common inhibitory neurotransmitter, glycine, in the retina as well, possibly directly (Neal et al. 1998). NO has effects on the heterologous gap junctions between AII amacrine cells and cone bipolar cells in the mammalian retina (Xin and Bloomfield 1999) where the AII is the archetypical glycinergic amacrine cell type. In the mammalian retina, GABAergic amacrine cells are usually wide-field cells (Vaney 1990; Kolb 1997), whereas the glycinergic ones are generally small-field cells with diffusely branching dendrites (Kolb 1997; Menger et al. 1998). NADPH-d-stained/nNOS-IR amacrine cells in mammalian retinas are wide-field, monostratified types and all colocalize GABA as far as we know (Vaney and Young 1988; Oh et al. 1998). Widefield amacrine cells in other species are commonly connected by gap junctions to each other (Vaney 1994; Kolb 1997), so the presence of NO in such cells would suggest a comparable role in receptive field modulation in all species’ retinas. NO acts on cyclic GMP to block gap junctions, so uncoupling horizontal cells and probably
amacrine cells as well (Miyachi et al. 1990; Miyachi and Nishikawa 1994). The intracellular recordings of the putative nNOS/GABA-IR cells that we have identified here are interestingly varied (Ammermüller et al. 1995; Ammermüller and Kolb 1995). A22 is an “Off” transient cell that shows distinct directional selectivity in its light response to moving targets. A15 is a sustained “On” center cell and A20 a transient On center cell. All three are likely to be connected into a syncytium across the retina by gap junctions between their processes. In contrast, glycinergic NADPH-d-stained amacrine cells have not been seen elsewhere but the turtle. What could be their function? The turtle NADPH-d glycinergic cells were small-field amacrine cells. Probably because of their tighter, multibranched dendritic trees, NO/glycinergic cells could influence visual processing in much more local circuits than possible for the wide-field GABAergic cells. The same could to be true for the chick retina, where 4 types of amacrine cells showed nNOS labeling and at least the small parvalbumin-IR cells are good candidates for being glycinergic (Fischer and Stell 1999). nNOS/glycine-IR amacrine cell types are candidate A1 and A6 types in the turtle retina, as mentioned above. Both have typical small-field amacrine cell intracellular responses, with complex sustained/transient components, or are On-Off in nature. Both A1 and A6 are also known to exhibit some color coding, with different signs of response to the two ends of the spectra (Ammermüller et al. 1995). In addition to amacrine cells, we detected a subset of nNOS-IR cells in the GCL (Fig. 5). Unfortunately, we were not able to decide whether these cells were nNOScontaining ganglion cells or displaced amacrine cells. In the literature, both can be found. In cat and rat, for example, NADPH-d labeling in the GCL has been attributed to displaced amacrine cells (Wässle et al. 1987; Yamamoto et al. 1993), whereas, in the turtle, NADPH-d activity was suggested to be in ganglion cells (Blute et al. 1997). The colocalization experiments we tried using nNOS with calcium binding proteins yielded positive immunostaining only to calbindin. Neither parvalbumin nor calretinin-IR cells showed nNOS at all. A subset of the GABAergic/nNOS amacrine cells were calbindin-IR. Colocalizations for other common neuroactive substances that are found in the retina (e.g., choline acetyltransferase, TOH, and somatostatin) failed. Colocalization with serotonergic amacrine cells and glucagon-synthesizing amacrine cells has yet to be tested. The serotonergic amacrines described by Weiler and Schütte (1985) show morphological similarities to some of the NADPHd-stained cells we saw here. We shall have to await further studies in this area. Acknowledgements We are grateful to D. Pow for providing antibodies, and to W. D. Eldred and H. Wässle for supportive comments.
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