Expression of neuronal markers, synaptic proteins, and glutamine synthetase in the control and regenerating lizard visual system

Abstract

Spontaneous regrowth of retinal ganglion cell (RGC) axons occurs after optic nerve (ON) transection in the lizard Gallotia galloti. To gain more insight into this event we performed an immunohistochemical study on selected neuron and glial markers, which proved useful for analyzing the axonal regrowth process in different regeneration models. In the control lizards, RGCs were beta-III tubulin- (Tuj1) and HuCD-positive. The vesicular glutamate transporter-1 (VGLUT1) preferentially stained RGCs and glial somata rather than synaptic layers. In contrast, SV2 and vesicular GABA/glycine transporter (VGAT) labeling was restricted to both plexiform layers. Strikingly, the strong expression of glutamine synthetase (GS) in both Müller glia processes and macroglial somata revealed a high glutamate metabolism along the visual system. Upregulation of Tuj1 and HuCD in the surviving RGCs was observed at all the timepoints studied (1, 3, 6, 9, and 12 months postlesion). The significant rise of Tuj1 in the optic nerve head and optic tract (OTr) by 1 and 6 months postlesion, respectively, suggests an increase of the beta-III tubulin transport and incorporation into newly formed axons. Persistent Tuj1(+) and SV2(+) puncta and swellings were abnormally observed in putative degenerating/dystrophic fibers. Unexpectedly, neuron-like cells of obscure significance were identified in the control and regenerating ON-OTr. We conclude that: 1) the persistent upregulation of Tuj1 and HuCD favors the long-lasting axonal regrowth process; 2) the latter succeeded despite the ectopia and dystrophy of some regrowing fibers; and 3) maintenance of the glutamate-glutamine cycle contributes to the homeostasis and plasticity of the system.

Expression of BDNF and NT‐3 during the ontogeny and regeneration of the lacertidian (Gallotia galloti) visual system

“…We found in addition abundant SMI-31 + cell nuclei, what is consistent with the putative nuclear lamin-B staining reported in different vertebrates (Weigum et al, 2003). Moreover, our data demonstrated the absence of BDNF within SV2 + + puncta and swellings were observed in putative degenerating/dystrophic fibers (Romero-Alemán et al, 2010). Conversely, in G. galloti NT-3 did not exhibit any punctate staining indicative of secretorygranule transport (Santos et al, 2008), which implies differential sorting and/or release mechanisms for different neurotrophins within the same system.…”

“…In fact, axotomy caused an apparent weak SV2 staining in the superficial tectum which persisted after reinnervation. In addition, putative regrowing fibers showed BDNF + and SV2 + puncta (Romero-Alemán et al, 2010). This labeling may represent intra-axonal transport which has been described both in developing and regenerating CNS axons (Bergmann et al, 2000;Lessman and Brigadski, 2009).…”

Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors

“…To study the status of newly generated neurons after lesion in detail, we examined the expression of established mature neuronal markers, such as MAP2a+b, parvalbumin, SV2 and metabotropic glutamate receptor 2 (mGlu2) (Buckley and Kelly, 1985;Kamiya et al, 1996;Kawaguchi et al, 1987;Pernet and Di Polo, 2006;Przyborski and Cambray-Deakin, 1995;Puyal et al, 2002;Reimer et al, 2008;Romero-Aleman et al, 2010;Scanziani et al, 1997;Yang et al, 2002). In the stab-lesioned zebrafish telencephalon, periventricular and parenchymal neurons derived from RG express MAP2a+b and parvalbumin (Fig.…”

Ontogeny of the conus papillaris of the lizard Gallotia galloti and cellular response following transection of the optic nerve: an immunohistochemical and ultrastructural study

“…The antibodies against GS, Pax2, GFAP and Tuj1 have been previously characterised for G. galloti by Western blot and negative controls (Romero-Alemán et al 2010). The anti-GFAP antibody was raised against a GFAP preparation from the pig spinal cord and recognises a single protein band between 51 kDa and 55 kDa in Western blots of a wide variety of vertebrates.…”

“…In addition, we have used the proliferating cell nuclear antigen (PCNA; Bravo and McDonald-Bravo 1987) to monitor the proliferative events in the CP. Moreover, in order to study the innervation of the CP of G. galloti, we have used an antibeta-III tubulin (Tuj1) antibody as a specific marker of postmitotic neurons (Moody et al 1989), as has also been employed to study ganglion cells in chick (Snow and Robson 1995), and an antibody against SV2 as a pansynaptic vesicles marker that is highly conserved among vertebrate species (Buckley and Kelly 1985;Okada et al 1994;Romero-Alemán et al 2010). Finally, conal cells and innervation have been examined by electron microscopy to complement the immunohistochemistry data.…”