Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians

Brain Research 926 (2002) 86–93 www.elsevier.com / locate / bres

Research report

Presence of thyrotropin-releasing-hormone-immunoreactive (TRHir) amacrine cells in the retina of anuran and urodele amphibians ´ Anadon ´ a , *, Marıa ´ Luz Dıaz ´ b , Manuela Becerra a , Marıa ´ Jesus ´ Manso b Ramon a

Department of Fundamental Biology, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain b ˜ , 15071 A Coruna ˜ , Spain Department of Cell and Molecular Biology, University of A Coruna Accepted 22 November 2001

Abstract The presence of thyrotropin-releasing-hormone-immunoreactive (TRH-ir) amacrine cells in the retina of amphibians is reported for the first time. The anuran and urodele retinas studied exhibit major differences in the distribution of TRH-ir cells. In the two urodele species investigated, most TRH-ir amacrine cells were located in the ganglion cell layer (GCL). These pear-shaped cells originate a dense TRH-ir dendritic plexus in strata 4–5 of the inner plexiform layer (IPL). A small number of TRH-ir amacrine cells were observed in the inner nuclear layer (INL). Most of these INL TRH-ir cells were multipolar neurons with radiating dendrites that originate a loose plexus in the IPL stratum 1. In the three anuran species investigated, most TRH-ir amacrine cells were located in the INL. Distribution of TRH-ir processes in the IPL of anurans was not so clearly layered as in urodeles, dendrites being observed throughout strata 1–5. In the toad retina THR-ir material was also observed in the outer plexiform layer, which suggests that toads may have some TRH-ir interplexiform neurons. In the frog and toad, TRH-ir fibers were also observed in the optic nerve, although their origin could not be ascertained. The number of TRH-ir amacrine cells per whole retina was higher in anurans than in urodeles, though urodeles have higher cell densities. The marked differences in distribution of TRH-ir amacrine cells observed between anurans and urodeles, and among the three anuran species, suggest different functions of TRH in retinal processing, perhaps related to the different specializations of the visual systems of these species.  2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Retina and photoreceptors Keywords: Thyrotropin-releasing hormone; Amacrine cell; Ganglion cell; Frog; Newt; Amphibian

1. Introduction The retina of amphibians, like that of other vertebrates, is composed of five main types of neurons: photoreceptors, bipolar cells, ganglion cells, horizontal cells and amacrine cells. Within these types, several subtypes can be distinguished. Different amacrine cell subtypes can be distinguished in Golgi-stained frog retinas by their morphology [35]. The retina of newts is thought to have an organization similar to that of frogs [13,21], although Golgi studies have not been reported. Amacrine cells represent a highly heterogeneous cell population as regards morphology, neurochemistry and electrophysiological *Corresponding author. Tel.: 134-981-563100x13295; fax: 134-981596904. ´ E-mail address: [email protected] (R. Anadon).

properties [11,12]. As in other vertebrates, immunocytochemical and autoradiographic methods have revealed a number of neurotransmitters and neuropeptides in the amacrine cells of amphibians [3,19,20]. In anuran retinas, previous studies have detected amacrine cells immunoreactive to GABA, glycine, serotonin, dopamine, dopamine-synthesizing enzymes, substance P, neuropeptide Y (NPY), somatostatin and CCK [19,20,23–25,41,46,51,55– 59]. In urodele retinas, previous studies have detected amacrine cells immunoreactive to GABA, glycine, serotonin, dopamine, glucagon, metenkephalin, NPY, vasoactive intestinal peptide (VIP), substance P, bombesin and neurotensin [1,8,9,19,20,30,47,48,50,52–54]. The retinas of the mudpuppy (Necturus maculosus) and other urodeles have been used for studies on the electrical activity of retinal cells, including amacrine cells [12,45,52]. Thyrotropin-releasing hormone (TRH), one of the first

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neuropeptides whose chemical structure was elucidated [22,39], is present in the retina [14,18,31,38]. Marked differences in retinal TRH concentration have been reported in different vertebrate groups [14]: the retinas of the frog (anuran) and eel (teleost) contain the highest concentrations of TRH. On the basis of the effects of retinotoxic compounds in the frog, it has been assumed that the TRH-containing cells are located in the inner retina [14], but until recently there has been no direct information available on which types of vertebrate retina cells contain TRH [7]. A recent immunocytochemical study in a teleost (trout), has shown the presence of TRH-immunoreactive (TRH-ir) amacrine cells in retinas of adults and of developing stages [2]. Here, we report for the first time the presence and distribution of TRH-ir amacrine cells in the retina of species representative of the two major extant amphibian groups, the urodeles and anurans.

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retinas were cryoprotected, frozen with liquid nitrogen, thawed three times and incubated as above except that the time of incubation with the TRH antiserum was increased to 3 days, and the time of incubation with the secondary and tertiary antibodies to 1 day each. The TRH antiserum (8940-0510-serum, batch AR981201C, Biogenesis) was raised in rabbit against TRH conjugated to haemocyanin by the dinitrofluorobenzene method. The specificity of the immunostaining was tested either by replacement of the primary antiserum with immune goat serum alone or by preabsorption of the primary antiserum, diluted 1:2000 in TBS, with synthetic TRH (Sigma, 50 mg / ml; 0.13 mM). These control experiments completely abolished immunostaining in the retina.

3. Results

3.1. Urodeles 2. Material and methods Adults of Bosca’s newt (Triturus boscai; seven specimens), salamander (Salamandra salamandra; two specimens), toad (Bufo bufo; two specimens), frog (Rana iberica; two specimens) and Iberian painted frog (Discoglossus galganoi; seven specimens) were caught in ´ del Galicia (Spain) with official permission (Consellerıa Medio Ambiente, Xunta de Galicia). The specimens were deeply anaesthetized with ethyl ether and fixed by vascular perfusion with cold 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After perfusion, their eyes were extracted and left in cold fixative (4 8C) for 24 h. Most retinas were cryoprotected in 30% sucrose in PB, frozen with liquid-nitrogen-cooled isopentane, and cut (10–12 mm thick) on a cryostat. Retinas of some individuals previously maintained in the dark for several hours were dissected out, whole-stained by the immunocytochemistry procedure and flat mounted. All procedures conformed to the European Community guidelines on animal experimentation. For immunocytochemistry, sections were treated with 3% H 2 O 2 and 10% methanol in 0.05 M Trizma-buffered saline (TBS) pH 7.6, for 30 min. The sections were then sequentially incubated at room temperature with: (1) two washes in TBS, followed by a wash in 0.1% Triton X-100 in TBS (TBS-T: 20 min); (2) normal goat serum (Dakopatts, Glostrup, Denmark; diluted 1:10 in TBS-T) for 1 h; (3) TRH antiserum (Biogenesis, Poole, UK; diluted 1:1000–1:2500 in TBS-T) for 20 h; (4) two washes in TBS-T; (5) biotinylated goat anti-rabbit (Dakopatts; 1:500 in TBS-T) for 1 h; (6) two washes in TBS-T; (7) StreptABComplex-horseradish peroxidase (Dakopatts) for 1 h; (8) 0.005 M Tris–HCl buffer pH 7.4 (2310 min). Finally, the immune complex was visualized by incubation with 0.005% diaminobenzidine (DAB, Sigma) and 0.003% H 2 O 2 in Tris–HCl for 10 min. For whole retina staining,

In the newt retina, two types of TRH-ir amacrine cells were observed. Most TRH-ir cells were located in the ganglion cell layer (GCL) and exhibited processes directed to the inner part of the inner plexiform layer (IPL) (Fig. 1A,B). TRH-ir cells of the GCL had one or two thick dendrites that were abundantly branched and exhibited a varicose and tortuous appearance. The arbors of contiguous cells overlapped, as seen in horizontal sections (Fig. 1B). These processes formed part of the two close dense TRH-ir plexuses of the inner third of the IPL, as seen in vertical sections through the retina (strata 4 and 5). A few TRH-ir cells were observed in the inner nuclear layer (INL), and most of their dendrites ran in the outer region of the IPL (stratum 1) (Fig. 1C). In horizontal or oblique sections, the TRH-ir cells of the INL were rather scarce and exhibited a multipolar appearance (Fig. 1D). Most of their dendrites ran straight over rather long distances and showed little branching, forming a loose plexus of varicose appearance next to the INL (Fig. 1D). Occasionally, processes of these INL cells were seen coursing to the inner TRH-ir plexuses (Fig. 1C), which suggests that some of these cells may be bistratified amacrine cells. Similar results were obtained in the retina of salamander, although the cells were smaller than in newt, and stratification of processes was less evident (Fig. 1E,F).

3.2. Anurans In the Iberian painted frog retina, TRH-ir amacrine cells were located only in the INL (Fig. 2A). Two morphologies of TRH-ir amacrine cells, diffuse and radiate, were sometimes distinguishable in sections, by the distribution of their dendrites. These amacrine cells had pear-shaped or oval-shaped perikarya and they gave rise to dendrites running horizontally in the IPL stratum 1 (radiate cells), and / or to dendrites running deeper and branching in strata

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Fig. 1. Sections through the retinas of Bosca’s newt (A–D) and salamander (E, F) showing TRH-immunoreactive amacrine cells. (A) Vertical section through the inner retina showing an amacrine cell of the CGL. (B) Photomontage of a tangential section through the inner retina showing processes and perikarya of amacrine cells of the CGL. (C) Vertical section through the inner retina showing an amacrine cell of the INL. (D) Photomontage of an oblique section through the inner retina showing long processes of an amacrine cell of the INL coursing in the stratum 1 of the IPL. (E, F) Vertical sections through the inner retina showing amacrine cells in the CGL and INL, respectively. Arrowheads, horizontal processes coursing in the IPL stratum 1. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bars, 25 mm.

2 and 3–5 (diffuse cells). In stratum 1, TRH-ir dendrites appeared clearly stratified, while those of strata 3–5 showed a ‘diffuse’ distribution. However, whether a TRHir cell can give rise to both types of dendrite (stratified and diffuse), or whether these two types of dendrite correspond

to two different types of TRH-ir cell (as in newt), could not be assessed. As in the newt retina, TRH-ir dendrites were varicose. In appropriately oriented sections, dendrites running in sublayer 1 could be followed over rather long distances. In the retinas of toad and frog, most TRH-ir

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Fig. 2. (A–D) Comparative vertical sections through anuran retinas showing TRH-ir perikarya in the INL. The retinas are from painted frog (A), Iberian frog (B, C) and toad (D). Note long horizontal processes in the stratum 1 of the IPL (arrows in A–C), and the presence of TRH-ir material in the outer plexiform layer (OPL) of the toad retina (D). Scale bars, A–D525 mm.

amacrine cells were located in the INL (Figs. 2B–D and 3E,F). A few cells were observed in the GCL of toad (Fig. 3A) and frog. Most TRH-ir processes were observed in the inner (3–5; most abundantly in the sublayer 3) strata of the IPL, but long horizontal processes were also seen in strata 1–2 (Figs. 2B,C and 3B). Characteristically, in the toad retina the outer plexiform layer (OPL) exhibited TRH-ir material (Figs. 2D and 3A–D). Although the connections of this OPL material with the TRH-ir cells of the INL were not clearly resolved, in some instances very thin processes were observed coursing between INL cells and the TRH-ir material of the OPL (Fig. 3C). At high resolution, the OPL TRH-ir material appeared to consist of very thin processes (Fig. 3D). In addition to cells located in the INL and GCL, some TRH-ir fibers were observed in the optic nerve and optic fiber layer of the frog and toad. In the optic nerve, these fibers were thin and varicose (Fig. 3E). About 100 TRH-ir fibers could be counted in a transverse section of the optic nerve of frog. In the Iberian painted frog and in urodeles, one or two fibers, could in some cases be seen crossing the optic nerve papilla, but they could not be followed into the

nerve. In the frog, perikarya of the GCL were contacted by TRH-ir processes and sometimes exhibited a moderate (ground?) stain (Fig. 2B). Whether the TRH-ir fibers observed in the optic nerves of the frog and toad arose from cells of the inner retina (ganglion cells) or from cells located in the brain could not be assessed.

3.3. Number of TRH-ir cells The estimated density of TRH-ir amacrine cells in the GCL in flat-mounted Bosca’s newt retinas was 6516176 cells / mm 2 , and their total number per whole retina about 2000 cells. The TRH-ir amacrine cells of the INL were not well stained in whole retinas. However, counts of INL TRH-ir cells in serial sections indicate that there are about 50 cells per whole Bosca’s newt retina. In the Iberian painted frog, the density of TRH-ir neurons estimated from tangential sections of the retina was about 3806132 cells / mm 2 and their total number per whole retina was about 4800 cells. For the retinas of the toad, the frog and the salamander the number of TRH-ir amacrine cells was

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Fig. 3. Vertical sections of the toad retina (A–D) and of the optic papilla of an Iberian frog (E). Note the different appearance of TRH-ir perikarya of the ganglion cell layer (A) and inner nuclear layer (B) and the presence of TRH-ir material in the outer plexiform layer (OPL) of the toad retina. The arrows in (B) point to the horizontal processes coursing near the INL, the arrowhead in (C) points to an outer process of a putative interplexiform neuron, and the outlined arrow in D points to fine TRH-ir processes of the OPL. (E) Section showing TRH-ir fibers (arrowheads) coursing in the optic nerve. Scale bars, A–D, 10 mm; E, 25 mm.

estimated on the base of direct counts in series of regularly spaced vertical sections of the whole retina. In this case, the number was corrected using Abercrombie’s factor, owing to the similar size and simple shape of perikarya. The retina of the toad had 12 40863594 TRH-ir cells (less than 1% of them located in the GCL), that of the frog contained 13 07963251 cells (about 1.5% of them located in the GCL), and that of the salamander 192061009 cells (about 79% of them located in the GCL). Taken together, these results indicate that anurans have more TRH-ir cells in the retina than urodeles, although urodeles have higher cell densities.

4. Discussion This study provides the first description of the TRH-ir cell populations of amphibian retinas. The anti-TRH antiserum also stains neurons and fibers throughout the retina and brain of trout [2,10]. As in the trout studies, preabsorption of the primary antiserum with synthetic TRH completely abolished immunostaining in amphibian retinas. These observations, together with the previous demonstration of high levels of TRH in the frog retina [14], strongly suggest that the substance presently demonstrated in amphibian retinas is TRH.

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Our results, together with recent evidence obtained in the retina of teleosts [2], appear to indicate that in the retina of vertebrates TRH is contained in amacrine cells. The number of TRH-ir cells estimated for all amphibian species appears to be much higher in amphibians than in trout, also in agreement with the finding that the frog retina contains the highest TRH concentration among retinas of species of several groups of vertebrates, including a teleost [14]. In urodeles, the presence of at least two different TRH-ir cell populations in the retina is clearly indicated by their localization, cell density and characteristics of their processes. Although our observations suggest that the same may occur in all anurans, present evidence is inconclusive in this respect. During development of the retina in trout, two different TRH-ir cell populations, monostratified and multistratified, could also be distinguished, though in adults only multistratified amacrine cells were observed [2]. Extensive overlap of TRH-ir processes in the IPL makes it difficult to compare the TRH-ir cell types observed in the retinas of the amphibian species with those obtained by other authors with Golgi methods or Lucifer yellow injected neurons. However, comparison of cell characteristics and density and / or number of TRH-ir cells with those reported for other peptide-containing amacrine cells in the retinas of newts [8,19] and frogs [23–25] suggest that TRH-ir cells are a new type(s) of peptide-containing amacrine cell. Only somatostatin-immunoreactive cells in the Xenopus retina exhibit a similar density, though these cells are interplexiform neurons [41], unlike the TRH-ir cells of urodeles and most anurans. Moreover, the number of TRH-ir cells and, in the case of urodele retinas, their localization is notably different from that of amacrine and / or interplexiform cells containing serotonin [19,20,49,39,53,56] or dopamine [48,51,57]. In urodele retinas, in addition, most TRH-ir cells are located in the GCL, a layer in which there have also been reports of a few displaced amacrine cells containing serotonin [8,19,53] or dopamine [48]. GABAergic displaced amacrine cells, however, were numerous in urodele retinas, often outnumbering the GABA-ir cells of the INL [9,19]. Although the GABAergic cells of the GCL were first considered as ganglion cells [8], studies in urodeles have provided strong evidence that they are amacrine cells [19]. Our results indicating the presence of a large number of displaced TRH-ir cells in the retinas of the two urodeles, and the absence of TRH-ir fibers in their optic nerves, support this hypothesis. Moreover, the abundance of TRHir cells and GABA-ir cells observed in the GCL of Bosca’s newt and salamander raises the possibility of co-localization of the two substances in amacrine cells. In Xenopus, most bipolar cell axons are confined either to strata 1–2 (the OFF zone) or 3–5 (the ON zone) of the IPL [42,51], conforming to Famiglietti and Kolb’s segregation pattern [15]. A similar segregation pattern of bipolar

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cell terminals is also probable for the amphibian species studied here. The presence of TRH-ir processes in both the distal (stratum 1) and proximal (strata 3–5) IPL, in all the amphibian species studied, suggests that the TRH-ir plexuses play modulatory roles in both the OFF and the ON zones, respectively, as suggested for other amacrine cells [51]. In the urodeles, the cells giving rise to the distal TRH-ir plexus likewise appear to be clearly different from those originating proximal processes. Moreover, the TRHir cells of the INL of urodeles are very scarce and show radial dendrites in the stratum 1, suggesting that these cells have wide receptor fields, while those of the GCL cells are abundantly branched and probably have smaller fields. Our observations reveal specialization of TRH-ir amacrine cells in two different retinal circuits. Although this is probably also the case in anurans, we were unable to confirm the existence of different populations in this group. The OPL of toad retina, unlike those of the other amphibians studied, shows TRH-ir material that appears to consist of very thin processes. This raises the possibility that some TRH-ir cells in the toad are TRH-ir interplexiform neurons, like some of the somatostatin- and dopamine-ir cells of the retina of Xenopus [40,41]. Finally, the presence of TRH-ir fibers in the optic nerve of Rana and toad indicates that they are retinofugal (optic fibers) and / or retinopetal axons. In Rana, peptide-containing ganglion cells have already been observed [27,28], and a small proportion of ganglion cells are GABA-ir [17]. Together with these findings, our results suggest that TRHir optic nerve fibers may originate from some retinal ganglion cells. However, the retina of the frog receives centrifugal fibers. Some retinopetal perikarya in Rana have been located in the lamina terminalis [44]. Moreover, horseradish peroxidase applied to the optic nerve labeled neurons in the optic tectum, although this labeling was attributed to transganglionic transport [26]. Since cells expressing TRH-like messenger are widely distributed in the anuran brain (Xenopus), including the forebrain and the optic tectum [60], to rule out the possibility that the TRH-ir fibers of the optic nerve are centrifugal axons needs further investigation. Despite the early demonstration of the presence of TRH in the retina [14,31,38], very little is known about its functions. In mammal pituitary and brain, TRH has been found to modify Ca 21 and K 1 channel gating kinetics through G-protein-mediated TRH receptors [4,5,16,29,36] and to down-regulate channel gene expression [43], producing short- and medium-term modifications of the membrane potential of adenohypophyseal cells and neurons. The presence of TRH receptors has been reported in the rat retina [32,34,37], and several neurotransmitters appear to induce release of TRH in the rat retina in vitro [33]. Moreover, a differential action of TRH on cat retinal (X and Y) ganglion cells has been observed in vivo: under photopic conditions the maintained activity and light response were suppressed in ON-centre cells and enhanced

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in OFF-centre cells, while under scotopic conditions TRH did not influence their discharge, suggesting a possible functional role of TRH in light adaptation [6]. Whereas the cellular location of TRH in mammalian retinas is currently unknown, our immunocytochemical results in teleosts [2] and in amphibians reveal a complex organization of TRHir amacrine cells in anamniote vertebrates, with at least two different populations recognizable in urodeles. Since these cells make contacts mainly in the ON and / or the OFF sublayers of the IPL, respectively, the precise modulatory effects of the TRH-ir cells on retinal output are probably different between the ON and the OFF circuits. Moreover, our results suggest the existence of important differences between urodeles and anurans, and among the three anuran species studied (each of which belongs to one of the three main extant orders of anurans). It may be suggested that the differences in TRH distribution among the retinas of these groups reflect adaptations to ensure optimal modulation of ganglion cells under different illumination conditions: frogs are largely diurnal, toads and urodeles largely nocturnal, and trout largely crepuscular. Further studies of TRH-ir amacrine cell populations of amphibians and fishes may shed additional light on the evolution of the TRH-mediated circuits of the retina.

Acknowledgements This work was supported by grants from the Spanish Science and Technology Ministry (BXX2000-0453-C0201 and -02) and the Xunta de Galicia (PGIDT99BIO20002).

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