interaction with cortical neurons, because muscimol treatment inhibits cortical neurons but does not prevent synaptic transmission itself. For example, it was reported that insufficient activation of cortical neurons can induce the depression of synaptic transmission [11], which may lead to afferent retraction through a retrograde message. To determine whether local synaptic interaction underlies the axon retraction in the inhibited cortex, it would be useful to examine the effect of cortical inactivation by blocking synaptic inputs instead of applying a GABAA receptor agonist. Botulinum neurotoxins are clostridial proteases that enter nerve terminals and act on the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins. Botulinum neurotoxin type E (BoNT/E) specifically cleaves synaptosomal- associated protein-25 kDa (SNAP-25) and blocks transmitter release [12,13]. Previous experiments demonstrated that BoNT/ E injections can produce a long-term inactivation of the visual cortex and hippocampus by blocking spontaneous and evoked neuronal activity [14,15]. Here, we inactivated the primary visual cortex of kittens by blocking synaptic transmission with cortical BoNT/E injections and examined the geniculocortical axon morphology in the animals with normal vision and those deprived of vision binocularly. We found that the afferent axons in the animals with normal vision show a significant retraction in the inactivated cortex, as similarly observed in the muscimol-treated cortex, whereas the axons in the binocularly deprived (BD) animals were mostly preserved. Therefore, the experience-driven axon retraction in the inactivated cortex can proceed in the absence of synaptic interaction. Results Functional effects of BoNT/E injection on visual cortex We first examined the effect of cortical BoNT/E injection on SNAP-25 and neuronal activity in the rat visual cortex. To estimate the duration of BoNT/E effects, we analyzed the time course of SNAP-25 cleavage after a single BoNT/E injection into the visual cortex. The visual cortical tissue was harvested on various days (1 day, 1 week, 2 weeks, and 4 weeks) after the injection of BoNT/E solution and subjected to western blot analysis using a monoclonal antibody that recognizes both intact and BoNT/E-cleaved SNAP-25. SNAP-25 cleavage was clearly detected 1 day and 1 week after the BoNT/E injection (Fig. 1). Cleaved SNAP-25 level decreased to undetectable level 2 and 4 weeks after the injection. Therefore, SNAP-25 cleavage in the visual cortex would persist for at least 1 week following BoNT/E Retraction of Active Axons injection. BoNT/E-injected animals did not show any sign of systemic intoxication during this time window. To determine the effect of BoNT/E injection on cortical activity, we recorded multiunit activity from the visual cortex of rats 1 week after BoNT/E injection. As shown in Fig. 2A, we did not observe any spike activity in the visual cortex treated with BoNT/E, whereas we found vigorous visual responses and spontaneous activity in the untreated visual cortex contralateral to the BoNT/E injection site. We further confirmed the blockade of neuronal activity in the BoNT/E-treated cortex by evaluating Egr-1 expression (Fig. 2B). The inducible expression of Egr-1 is commonly used as a marker of neuronal activity [16]. The level of Egr-1 expression significantly decreased in the BoNT/E-treated cortex compared with that in the cortex of normal animals (average6SEM: 43.561.2%, p,0.05, unpaired t-test). Thus, single BoNT/E treatment could effectively block neuronal activity in the visual cortex at least for 1 week. Effects of visual experience on arborization of geniculocortical axons in BoNT/E-treated cortex We labeled geniculocortical afferents of 6-week-old kittens by microinjecting an anterograde tracer, biotinylated dextran amine (BDA), into lamina A of LGN. Activity of the visual cortex was suppressed by disrupting synaptic transmission by cortical injection of BoNT/E. A group of kittens was deprived of vision in both eyes by eyelid suture. After 1 week, the cortical region inactivated by BoNT/ E was delineated physiologically by mapping the activity of cortical cells. As similarly observed in rats (Fig. 2A), we found no spontaneous or visually evoked activity in the region around the BoNT/E injection sites. The inactivated area extended up to 10 mm in the anterior-posterior direction. We further confirmed the cortical inactivation by Egr-1 immunostaining (Fig. 3). Egr-1-immunoposi- tive cells were distributed at a high density in the area far from the injection sites (Figs. 3A and B). In contrast, the density of Egr-1- immunopositive cells markedly decreased in the area near the injection sites (Figs. 3A and C). The region of very low Egr-1 immunoreactivity included the entire medial bank and often extended beyond the sulcus suprasplenialis and the sulcus lateralis. Cortical arbors of geniculocortical axons in the BoNT/E inactivated area were labeled well with BDA, as shown in Fig. 3D. We found no difference in the quality of labeling between the axons in BD animals and those in animals with normal vision. In this study, 10 and 9 axonal arbors in the inactivated region of area 17 were reconstructed from the normal-vision animals (n = 5) Figure 1. Cleavage of SNAP-25 by BoNT/E injection in visual cortex. (A) Representative immunoblots for SNAP-25 in rat visual cortex on various days after BoNT/E injection. Cleaved SNAP-25 with a lower molecular weight is clearly observed 1 day and 1 week after BoNT/E injection (upper panel). Norm, normal animals. (B) Quantification of cleaved SNAP-25. Blot densities were normalized to those of GAPDH. Error bars indicate SEM. *: P,0.05 (n = 3–5 each, one-way ANOVA followed by post hoc Scheffe test). doi:10.1371/journal.pone.0004193.g001 PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4193