distance and accessing movement of two fish (Kato et al. 2004). We measured changes of chasing behavior for 30 min at various time points after optic nerve injury by using an image processing system. In control pair, the chasing ratio was 72% (Fig. 12.2b, 0 day). The bilateral optic nerve transection of two fish induced a significant reduction of this ratio to 20% just after axotomy. The low value was maintained for 1–2 weeks and then the chasing ratio gradually increased 40% at 40 days after axotomy. The value (40%) continued during 50–120 days and then was slowly recovered to control value by 180–200 days after injury (Fig. 12.2b).

12.4 Discussion

12.4.1 Termination of Optic Nerve Regeneration in Goldfish

The optic nerve regeneration in goldfish is a long process and therefore it is very difficult to decide the endpoint of this process. As for the fish optic nerve regeneration process, the excess number of ectopic optic axons initially reinnervate the tectum 3–6 weeks and then the optic axons are repulsed 2–4 months after axotomy. Finally exact topographic retinotectal connections are completed 5–6 months after axotomy (Edwards et al. 1985; Meyer and Kageyama 1999). In a previous paper, we followed up the time course of cell body response to nerve injury in the goldfish retina for over 4 months (Kato et al. 1999). We found that cell body of RGCs after optic nerve transection became hypertrophic 1 week and peaked 2-folds at 2 months after axotomy. Then the cell body returned to the normal size by 4 months after axotomy (Kato et al. 1999). Moreover, we described that two-point distance of pair of goldfish with axotomized optic nerve was initially very long and then gradually shortened 1.5–4 months and finally recovered the control value 5–6 months after axotomy (Kato et al. 1999). From these morphological and behavioral observations, the finish of optic nerve regeneration in goldfish is more than 5–6 months after axotomy. So far as we know, we have no biochemical marker expressing throughout the whole period of optic nerve regeneration. It is worthy to search such a maker for announcing the initial and terminal point of this long process, in basic and clinical neuroscience of nerve regeneration. In this view point, the present study was performed.

12.4.2GAP43 Is a Good Marker for Monitoring the Long Process of Optic Nerve Regeneration in Fish

GAP43 protein was originally discovered as a rapidly transported acidic protein from cell bodies to the axons in the regenerating optic nerves after nerve crush (Skene and Willard 1981; Benowitz et al. 1981). Later, GAP43 was further localized at the major growth cone and presynaptic terminals (Deckker et al. 1989). In

p-Akt

p-Bad

Caspase-3

GAP43

Fig. 12.3 Time course of regeneration-associated molecules in expression after optic nerve transection in goldfish. IGF-I, p-Akt and p-Bad expression increased 1–5 days (Koriyama et al. 2007) after axotomy. Purpurin expression increased 2–5 days (Matsukawa et al. 2004) after axotomy. Transglutaminase (TGR) expression increased 10–30 days (Sugitani et al. 2006) after axotomy. Bcl-2 expression also increased 10–20 days and caspase-3 expression rather decreased in this period (Koriyama et al. 2007). In contrast, GAP43 expression increased for a long time 3–150 days after axotomy with a biphasic expression pattern

a previous paper (Bormann et al. 1998), changes of GAP43 mRNA were followed up in the zebrafish retina until 56 days after optic nerve transection. Their in situ hybridization studies showed that positive signals of GAP43 mRNA increased in the RGCs 1–2 weeks and then gradually decreased by 56 days after optic nerve injury. In the present study we investigated changes of GAP43 protein in the goldfish RGCs over 180 days after axotomy. The immunoreactivity of GAP43 started to significantly increase 5 days, peaked at 2–3 weeks and continued for near 150 days after axotomy (Fig. 12.1). Interestingly, the expression pattern of GAP43 mRNA was clearly biphasic, a short peak phase (9-folds) at 7–20 days and a long plateau phase (5-folds) at 50–120 days after axotomy (Fig. 12.2a). In the time course of schooling behavior of pair of goldfish with bilateral optic nerve transection was also biphasic, a rapid recover phase at 7–40 days and a long plateau phase at 50– 120 days after axotomy. The similar time course between GAP43 expression and recovery phase of chasing behavior leads to a conclusion that changes in GAP43 expression in the fish retina during optic nerve regeneration well reflect the state of visual function after nerve injury. We have cloned many factors and enzymes as a regeneration-associated molecule during goldfish optic nerve injury (Liu et al. 2002; Matsukawa et al. 2004; Sugitani et al. 2006; Koriyama et al. 2007), they almost all worked as an axonal elongation factor in the goldfish optic nerve regeneration system (Fig. 12.3). GAP43 is an exceptional molecule which works throughout the whole period of optic nerve regeneration with a characteristic biphasic expression pattern. Further experiment is needed to elucidate the role of GAP43 on the short peak (7–20 days) and the long plateau (50–120 days) phases in the regrowing optic axons.