Retraction of Active Axons Figure 2. Effects of BoNT/E on cortical activity and Egr-1 expression level in visual cortex. (A) Examples of spike activity in rat visual cortex contralateral (upper trace, Control) and ipsilateral (lower trace, BoNT/E) to BoNT/E injection site recorded 1 week after injection. BoNT/E blocked neuronal activity only in the injected cortex. (B) Egr-1 expression level is significantly decreased in the BoNT/E injected cortex. The upper right panel shows examples of immunoblots for Egr-1 in the visual cortex of normal rats (Norm) and that 1 week after BoNT/E injection. Blot densities were normalized to those of GAPDH and expressed as the ratio to those of Egr-1 in normal animals. Error bars indicate SEM. *: P,0.05 (n = 5 each, unpaired t-test). doi:10.1371/journal.pone.0004193.g002 and BD animals (n = 3) in three dimensions, respectively. Four examples of arbors from each group are shown in Fig. 4. All of the arbors in this study represent those serving the contralateral eye. The terminal arborizations of geniculocortical axons in both normal-vision and BD animals were mainly localized in layer IV. The arbors in the normal-vision animals showed a marked reduction in the complexity of terminal arborization compared with those in BD animals (Figs. 4A and B). In addition, the cortical area covered by each arbor in the normal-vision animals was much smaller than that in BD animals, as clearly shown in surface views of the arbors. We quantitatively determined the axonal morphology by measuring two parameters, the total length of axon segments and the number of branch points of the cortical arbors of individual geniculocortical axons and compared them with published results for the arbors in the animals under various experimental conditions (Fig. 5 and Table 1) [9,10,17]. In the BoNT/E-treated cortex, the arbors in the normal-vision animals were significantly shorter and had fewer branch points than those in the BD animals. Small arbors in the normal-vision animals should reflect the net shrinkage of arbors rather than the suppression of development because they were significantly smaller than those at the start of BoNT/E injection (normal P40 animals). On the other hand, the arbors in the BoNT/E-treated cortex of the BD animals were preserved and similar in branching number to those in the normal animals at the start of BoNT/E injection (normal P40), although they were somewhat shorter in total length. Thus, in the cortex inactivated by BoNT/E injection, the arbors serving the open eye, which carry visual information, showed a highly significant shrinkage and the shrinkage could be alleviated by visual deprivation, as observed in the cortex inhibited by muscimol treatment [9,10]. The open-eye arbors in the muscimol-treated cortex (MD-ND and Norm in Muscimol 2w, Fig. 5 and Table 1) showed a much more significant shrinkage than those observed in the BoNT/E- treated cortex. The more significant shrinkage in previous muscimol experiments should reflect the longer cortical inactiva- tion rather than a difference in pharmacological effects of BoNT/ E and muscimol, because muscimol treatment for 1 week induced the same level of shrinkage as BoNT/E treatment (MD-ND in Muscimol 1w, Fig. 5 and Table 1, unpublished observation). On the other hand, the arbors in the BoNT/E-treated cortex of BD animals were similar to the arbors serving the deprived eye in the muscimol-treated MD animals, although they were shorter than those in muscimol-BD animals (MD-D and BD in Muscimol 2w, Fig. 5 and Table 1). Therefore, experience-dependent axon retraction can occur in the absence of synaptic transmission when cortical activity is suppressed. PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4193