Brain Research 883 2000 98–106 www.elsevier.com locate bres
Research report
Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97
a b
a a
´ Almudena Velasco , Marıa Julia Bragado , David Jimeno , Elena Caminos ,
a a
a ,
´ ´
´ Concepcion Lillo , Jose Aijon , Juan M. Lara
a
´ ´
´ Instituto de Neurociencias de Castilla y Leon
, Departamento de Biologıa Celular y Patologıa, Universidad de Salamanca, E-37007 Salamanca, Spain
b
´ ´
´ Departamento de Fisiologıa
, Universidad de Alcala de Henares, Madrid, Spain Accepted 2 August 2000
Abstract
We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual
system tissues 105, 115, 160, 200, 325 and 335 kDa approximately. By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis
we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of
the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic
nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.
2000 Elsevier
Science B.V. All rights reserved.
Theme : Development and regeneration
Topic : Regeneration
Keywords : Fish; Neurofilaments; Optic nerve; Optic tectum; Retina
1. Introduction levels, in the axonal transport and in the cytoskeleton of
the ganglion cell axons [13,17]. The retinas of some teleosts have two peculiarities with
In relation to the cytoskeleton of fish retinal ganglion respect to other vertebrates: the retina grows throughout
cells, different studies of the expression of the proteins of the life of the animal from a peripheral germinal zone [26]
the neurofilaments, both in the normal state and after a continuously adding new neurons and axons of the gang-
lesion [9,14,22,29], have been carried out. In mammals, lion cells and the retinal ganglion cells are capable of
principal neurofilament proteins have been classified into regenerating their axons and restoring vision after a lesion
three types according to their molecular weight: neuro- in the optic nerve [4,17,31]. During the growth and
filament-light NF-L, neurofilament-medium NF-M and regeneration processes important changes occur in the
neurofilament-heavy NF-H [32]. In fish, differences in visual pathways, both at morphological and biochemical
the composition of these proteins have been described in comparison to those of mammals. The predominant pro-
teins of the neurofilaments of the goldfish optic nerve have a low molecular weight, such as gefiltin, a type IV
Corresponding author. Tel.: 134-923-294-500; fax: 134-923-294-
neurofilament protein [15] and plasticin, a type III neuro-
549. E-mail address
: rororogugu.usal.es J.M. Lara.
filament protein [14]. NF-M proteins have also been
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A . Velasco et al. Brain Research 883 2000 98 –106
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detected in the goldfish optic nerve with molecular weights with 3 H O in 100 methanol for 10 min. After rinsing
2 2
of 145 kDa [16,30]. However, NF-H proteins have not twice for 10 min in phosphate buffer saline solution with
been identified in the optic nerve of goldfish [13]. These 0.2 Triton X-100 PBS–Tx, the sections were incubated
differences with regard to the mammalian proteins appear overnight with RT97 monoclonal antibody Hybridoma
to be important in the continued plasticity of the visual Bank Iowa University at a dilution of 1:1000 at 188C in a
system [13]. Several of these proteins, such as plasticin humidity chamber, and subsequently incubated at room
and gefiltin, modify their expression during regeneration temperature with biotinylated mouse IgG Vector at a
[12,14,15,27,28,33]. Nevertheless, in the visual system of dilution of 1:200 and avidin–peroxidase complex Vector
mammals [5,10,36] and several anamniotes, such as 1:250. The sections were finally reacted with 0.025
Xenopus and trout [6,38], proteins of NF-H in the axon 3.39-diaminobenzidine
tetrahydrochloride DAB
and ganglion cells have been detected. These proteins are also
0.025 hydrogen peroxide in 0.2 M Tris–HCl pH 7.6 found in other areas of fish brains [1,9,22].
for 10 min at room temperature. Sections were dehydrated The antibody RT97 has been used to recognize the
and mounted in Entellan Merck. The right retinas and phosphorylated 200-kDa neurofilament sub-unit in mam-
optic nerves of lesioned tench and the visual pathways mals [37]. Furthermore it also recognizes, though with
retinas, optic nerves and optic tecta of unlesioned tench lesser affinity, another phosphorylated neurofilament pro-
were used as controls. Immunohistochemical controls were tein of 155 kDa and also tau of Alzheimer neurofibrillary
performed either by the omission of the primary antibody tangles [2,3,8,21] and the upper band of the MAP1B
or by replacement with a non-immune serum. doublet [19]. This antibody has also been used to label
specific populations of neurons in the rat [20,23]. In this 2.1. Immunoprecipitation Western blot
study, we analyze the distribution and modification of the immunostaining of RT97 and which proteins are recog-
2.1.1. Tissue preparation nized by this antibody in the normal and damaged tench
Retina, optic nerve and optic tectum from control and visual system.
lesioned tench 30 days post-crushing were dissected and isolated. Tissue was sonicated twice at 48C for 5 s in lysis
buffer which contained 50 mM Tris HCl, pH 7.4, 150 mM
2. Material and methods
