Retraction of Active Axons Figure 3. Example of labeled axons in BoNT/E-treated visual cortex. (A) Low-magnification image of coronal section across visual cortex of BoNT/E-injected kitten. An arrowhead indicates the BoNT/E injection site. (B, C) High-magnification view of two regions in A far from (dotted line, B) and close to (solid line, C) the injection site. Egr-1 signal is detectable in the area far from the injection site (B), whereas the signal is very low in the area near the injection site (C). The staining at the lower-left in C represents labeled axons. (D) Enlarged view of geniculocortical axons in BoNT/E inactivated area (box in C). Scale bar, A: 1 mm, B, C: 100 mm, D: 10 mm. doi:10.1371/journal.pone.0004193.g003 Discussion In this study, the geniculocortical axons in the visual cortex inactivated by BoNT/E treatment showed a highly significant shrinkage and the shrinkage could be alleviated by visual deprivation (Figs. 4 and 5, Table 1), as observed previously in the muscimol-treated cortex [9,10]. These results indicate that the axon retraction in the inactivated cortex can proceed in the absence of synaptic transmission. BoNT/E is a metalloprotease that selectively cleaves the synaptic protein SNAP-25 and prevents transmitter release [12,13]. Because administration of BoNT/E can block synaptic activity reversibly, it is used as a tool to address the role of synaptic activity in vivo [14,15]. We found no spontaneous or visually evoked activity in the BoNT/ E-treated region of the visual cortex even 1 week after the administration. Consistent with previous reports [14,15], we did not find a sign of general deleterious effect of BoNT/E on the cortical tissue. The activity blockade was well confined to the vicinity of the injection sites, which extended up to 10 mm in the anterior- posterior direction, and we observed vigorous visual responses outside the area and in the contralateral visual cortex. In addition, we detected small activities in the middle cortical depth, possibly representing afferent activity, as similarly observed in the muscimol- treated cortex [9,10,18], indicating that BoNT/E did not disturb the electric activity of afferent axons. The Nissl-stained sections of the BoNT/E-treated cortex showed no differences in neuronal density and cortical thickness from those of the normal cortex. Finally, if BoNT/E had exerted a deleterious effect on afferent axons, such effect should have caused a degradation of presynaptic afferents for both eyes. The present finding that the retraction of afferents in the BoNT/E-treated cortex depends on their own activity argues strongly against this possibility. Previous results in the muscimol-treated cortex suggest that presynaptic activity in the absence of postsynaptic activity leads to the retraction of afferent axons, on the basis of the assumption that muscimol selectively suppresses the activity of postsynaptic cells and does not have direct presynaptic effects. Pharmacological studies showed that muscimol binds selectively to GABAA receptors, which are located only on postsynaptic sites [19,20]. However, it was also reported that muscimol at a high concentration might act on GABAB receptors, which are located on geniculocortical axon terminals [21]. This finding raises a possibility that the retraction of open-eye axons observed in the muscimol-treated cortex might be induced by activation of presynaptic GABAB receptors rather than by cortical activity blockade. The present finding that open-eye afferents retract in the BoNT/E-treated cortex provides evidence against this possibility because BoNT/E can inactivate the visual cortex without activating GABAB receptors. The afferent arbors in the BoNT/E-treated cortex of normal- vision animals showed a significant retraction, because they were smaller than those at the start of BoNT/E injection (arbors in the normal P40 animals). The open-eye arbors in the cortex treated with muscimol for 1 week showed a retraction extent comparable to that in the BoNT/E-treated cortex (MD-ND in Muscimol 1w, Fig. 5 and Table 1). Thus, it is reasonable to state that cortical inhibition by BoNT/E induces the retraction of afferent axons to the same extent as cortical inactivation by muscimol. On the other hand, visual deprivation prevented afferent arbors from retracting in the BoNT/E-treated cortex, as found previously in the muscimol-treated cortex [10]. However, the deprived-eye arbors in the BoNT/E-treated cortex were much shorter in total length and had fewer branch points than the arbors in the normal animals at the time of perfusion (P50, Fig. 5 and Table 1), suggesting that cortical inactivation prevented an axonal elonga- tion during normal development. Furthermore, the deprived-eye arbors in the BoNT/E-treated cortex were shorter in length than the arbors in the normal animals at the time of BoNT/E injection PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4193