Early development of GABAergic cells of the retina in sharks: An immunohistochemical study with GABA and GAD antibodies

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Abstract

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27–29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.

Introduction

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter that participates in the encoding of sensory information in the vertebrate retina. It is thought that GABA plays a neurotransmitter role in interneurons involved in lateral pathways (amacrine cells, horizontal cells and interplexiform cells; for a review see Kalloniatis and Tomisich, 1999), although recently it has been proposed that in horizontal cells GABA also acts as a neuromodulator (Kreitzer et al., 2003, Kreitzer et al., 2007). GABA and its synthesizing enzyme glutamic acid decarboxylase (GAD) have been localized in amacrine cells in all vertebrates, but their presence in horizontal, interplexiform and ganglion cells is dependent on the species, the GAD isoform or the developmental stage. Knowledge of the GABAergic cells of the retina of elasmobranches is based on results of GABA and GAD immunohistochemical studies (Brandon, 1985, Brunken et al., 1986, Agardh et al., 1987), which have revealed the presence of GABAergic amacrine cells and, in some species, GABAergic horizontal and/or interplexiform cells. GABAergic horizontal cells have been reported in species of Raja (Agardh et al., 1987), whereas GABAergic interplexiform cells sending distal processes were reported in Raja erinacea and Mustelus (Brunken et al., 1986). In contrast with other vertebrates, no GABAergic cells were reported in the ganglion cell layer of elasmobranches. Two GABA transporters have been cloned in a skate and were shown to be located in horizontal and Müller cells (Malchow and Andersen, 2001, Birnbaum et al., 2005). The presence of GABA receptors and their responses to GABA have also been studied in some retinal cells of a skate (Malchow et al., 1989, Malchow and Ripps, 1990, Qian et al., 1996, Qian et al., 1997, Qian et al., 2001).

Other roles besides neurotransmission have been attributed to GABA in the retina, because it is one of the neuroactive substances that appears earlier during retinogenesis, prior to synaptogenesis. It has been proposed that GABA acts in the developing retina as a neurotrophic or signalling factor, promoting synaptogenesis, growth and differentiation of neurons (Versaux-Botteri et al., 1994, Sandell et al., 1994, Buznikov et al., 1996, Wang et al., 2007). However, the onset of the expression of GABA in retinal cells differs among vertebrates (Hokoç et al., 1990, Versaux-Botteri et al., 1994, Sandell et al., 1994, Negishi and Wagner, 1995, Hagedorn et al., 1998, Yamasaki et al., 1999, Nguyen and Grzywacz, 2000). Moreover, GABA has been implicated in the development and maturation of the outer retina because of its transient expression in horizontal cells (Schnitzer and Rusoff, 1984, Redburn and Madtes, 1987, Versaux-Botteri et al., 1989, Messersmith and Redburn, 1992, Messersmith and Redburn, 1993, Pow et al., 1994, Fletcher and Kalloniatis, 1997, Yamasaki et al., 1999, Dkhissi et al., 2001, Loeliger and Rees, 2005).

Studies on the development of retinal GABAergic cells have been carried out in some tetrapods (Yamasaki et al., 1999, Nguyen and Grzywacz, 2000, Dkhissi et al., 2001, Calaza et al., 2003, Loeliger and Rees, 2005), a few teleosts (Sandell et al., 1994, Negishi and Wagner, 1995, Hagedorn et al., 1998) and the sea lamprey (Anadón et al., 1998, Meléndez-Ferro et al., 2002, Villar-Cerviño et al., 2006). These studies revealed marked differences between agnathans and teleosts in the development of this neuronal system. Accordingly, the developmental pattern of the GABAergic cells in retinas of teleosts might not be representative of the early basic plan in jawed vertebrates. Cartilaginous fishes (Chondrichthyes) are the sister group of jawed vertebrates with a bony skeleton (Teleostomi) – which include land vertebrates – and thus are especially appropriate for out-group cladistic analysis. Moreover, the elasmobranch retina appears especially suitable for developmental studies because of its large size and slow development, which would facilitate detailed analysis. However, nothing is known about the neurochemical development of the elasmobranch retina, and the scarce data on its morphogenesis are derived from light microscopic studies with conventional staining methods (Yew, 1982) or from ultrastructural studies (Fishelson and Baranes, 1999). On the other hand, the pattern of expression of some developmental genes in the eye has been reported in very early stages of Scyliorhinus canicula (Sauka-Spengler et al., 2001, Plouhinec et al., 2005).

The main aim of the present study was to analyse how the GABAergic cells develop in the retina of elasmobranches, using immunocytochemistry with antibodies raised against two GABAergic markers, GABA and GAD. We studied the spatiotemporal pattern of retinal structures in embryos, juveniles and adults of the dogfish (S. canicula; Scyliorhinidae), a species that has been largely used as model for neuroanatomical studies. For comparative purpose, we have also analysed in parallel another member of the family Scyliorhinidae, the shyshark (Haploblepharus fuscus). This study revealed for the first time that the development of GABAergic neurons in the retina in these elasmobranches is remarkably unlike that of teleosts as regards their very early appearance, before any layering was settled in the retinal neuroepithelium. Moreover, the presence of GABAergic cells in the ganglion cell layer and of GABAergic fibres in the optic fibre layer and optic nerve, as well as the transient expression of GABAergic markers in the horizontal cell layer, are firstly reported in elasmobranches.

Section snippets

Experimental animals

Embryos and juvenile specimens of the lesser-spotted dogfish (S. canicula) and the brown shyshark (H. fuscus) were kindly provided by the “Aquário Vasco da Gama” and “Oceanário” in Lisbon (Portugal) and the Aquarium Finisterrae in A Coruña (Spain). The embryonic stages of dogfish were identified by external features according to Ballard et al. (1993) and similar features were considered in staging the shyshark embryos. The following embryonic stages were analysed: stage 24 (with diamond-shaped

Results

No differences were observed in the distribution of GAD immunoreactivity through the developing and mature retina of either species. Moreover, both GAD and GABA antibodies revealed the same pattern of immunoreactive (ir) cells in the embryonic retina of S. canicula (H. fuscus was not processed for GABA). In the following, we will describe the retina of both sharks together and to use the term GABAergic to refer to GAD- and GABA-immunoreactive structures.

Comparison of GABA and GAD expressions in the shark retina

In birds and mammals, the delayed onset of GAD expression with respect to that of GABA has led to the suggestion that in early developmental stages GABAergic retinal cells may synthesize GABA through a biochemical pathway that involves putrescine as a GABA precursor instead glutamate (Hokoç et al., 1990, Yamasaki et al., 1999). In the present work we showed that the earliest GAD signal was detected at the same stage that displayed GABA-immunoreactive cells (stage 26 embryos), thus suggesting

Acknowledgements

We thank F. Gil, G. Nunes and A. Cribeiro for their help in obtaining dogfish embryos. This work was supported by Spanish Science Ministry-FEDER (BFU2004-03313; BFU2007-61154/BFI) and Xunta de Galicia (PGIDIT05PXIC20003PN, PGIDIT06PXIB200065PR).

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