further reacted with secondary anti-mouse IgG (1:500). The immunoreactivity was visualized with avidin-biotin immunoenzymatic method.

12.2.3 RT-PCR Analysis

Primers of forward and reverse forms of goldfish GAP43 cDNA were determined by referring to that of goldfish GAP43 cDNA (database No. emb: M26250). RT-PCR was performed in a linear range of product. GAPDH mRNA levels were used as a control under non-saturation level.

12.2.4 Behavioral Analysis

Images of moving two goldfish with bilateral optic nerve transection were captured by a computer image processing system (Kato et al. 2004). The positional coordinates of moving two fish were treated and analyzed at a frame rate of 30/s. The chasing ratio of two fish was counted as a percentage of chasing times/total observation time (Kaneda et al. 2008).

12.3 Results

12.3.1Immunohistochemical Studies of GAP43 Protein

in the Goldfish Retina After Optic Nerve Transection

We followed up the changes of GAP43 protein in the goldfish retina until 180 days after nerve injury with an immunohistochemical analysis. In the control retina, a faintly positive signal of GAP43 protein could be seen in the RGCs (Fig. 12.1a). The positive signals of GAP43 slightly increased in the RGCs 5 days (Fig. 12.1b) and peaked at 20 days (Fig. 12.1c) after axotomy. Thereafter, the positive signals gradually decreased in the RGCs 60 days (Fig. 12.1d) and 90 days (Fig. 12.1e), and finally returned to the control levels by 180 days (Fig. 12.1f) after nerve lesion.

12.3.2Time Course of GAP43 mRNA Expression in the Goldfish Retina During Optic Nerve Regeneration

Next, we semi-quantitatively measured the level of GAP43 mRNA in the retina over 180 days after axotomy using RT-PCR method. After electrophoresis, a 639 bp single band was detected. The level of GAP43 mRNA rapidly increased at 3 days, peaked at 7–20 days (about 9-folds) and then rapidly decreased to 6-folds by 30 days after axotomy (Fig. 12.2a). The over control increase (5-folds) lasted for a long time (50–120 days), and then gradually returned to the basal level by 180

Fig. 12.1 Changes of GAP43 protein in the goldfish retina after optic nerve transection with immunohistochemical analysis. (a) In the control retina, weak immunoreactivity of GAP43 could be seen in the goldfish retinal ganglion cells (RGCs). (b) The immunoreactivity started to increase in the RGCs at 5 days (b), peaked at 20 days (c) and then gradually declined at 30–40 days after axotomy with a significant increase of immunoreactivity. Then the significant increase of immunoreactivity continued in the RGCs at 60 days (d) and gradually decreased at 90 days (e) and finally returned to the control value by 180 days after axotomy (f). Scale = 50 μm

days after axotomy (Fig. 12.2a). Thus, the time course of GAP43 mRNA levels in the retina after axotomy showed a biphasic pattern, a short peak increase and a long plateau increase (Fig. 12.2a). The PCR product was sequenced and the sequence was completely matched with that of goldfish cDNA fragment for GAP43. The levels of GAPDH mRNA did not change throughout the whole periods (data not shown).