development of sympathetic and olfactory neurons [23,24]. It is of interest to note that p75 was observed to be regulated in an activity-dependent manner in a study using neuronal cultures [25]. It is possible that such an activity-dependent regulation of repulsive interactions underlie the experience-driven axon retrac- tion in the visual system. Although such hypothetical presynaptic mechanism of axon retraction should operate also in the normal (active) cortex, the afferent axons serving the open eye of MD animals and those serving either eye in the normal-vision animals without muscimol infusion had much longer length and more branching than those in the inactivated cortex. When the cortex is active, cortical neurons may release some substance to rescue the axonal retraction or stabilize afferent synapses from the retraction. For example, neurotrophic factors are known to be released from target neurons and promote axonal growth [26,27]. Especially, previous studies demonstrated that brain-derived neurotrophic factor (BDNF) influence the cortical projection of LGN axons (ocular dominance columns) [28,29] and its expression is regulated in activity-dependent manner [30]. Therefore, two modes of plasticity which allow weakening and strengthening of particular pathways might operate concertedly in the developing visual cortex. In conclusion, the present findings provide anatomical evidence that the experience-driven axon retraction in the inactivated visual cortex can proceed in the absence of synaptic transmission. These results suggest that presynaptic mechanisms play an important role in the experience-dependent rearrangement of LGN axons. Materials and Methods All kittens (n = 8) in this study were born in the breeding colony of the Tottori University Research Center for Bioscience and Technology. All Long-Evans rats (n = 19) in this study were obtained from Shimizu Laboratory Animal Supply Co. Ltd (Kyoto, Japan). The experimental procedures used met the regulation of the animal care committee of the Tottori University. Surgery A 30 G stainless steel cannula connected to a 10 ml Hamilton syringe with a polyethylene tubing was inserted into the left hemisphere of area 17 of the visual cortex in 6-week-old kittens (postnatal days (P) 41–43). BoNT/E solution (100 nM in phosphate-buffered saline (PBS, pH 7.4) containing 2% rat serum albumin) was injected stereotaxically at three locations (A = 22.0 mm, L = 1.0 mm; A = 24.0 mm, L = 1.0 mm; A = 26.0 mm, L = 1.0 mm) at a depth of 2 mm from the cortical surface (2 ml at each location; rate, 0.2 ml/min). To label geniculocortical axons, we injected an anterograde tracer, biotinylated dextran amine (BDA, MW 10 kDa, Molecular Probe). A glass microelectrode filled with BDA solution (10% in 0.5 M NaCl) was positioned stereotaxically at LGN, which is ipsilateral to the BoNT/E injection sites, with verification of depth by monitoring visual responses. We used the pipettes with fine tips (5–10 mm diameter) to minimize tissue damage that might label the fiber of passage. BDA was injected iontophoretically (pipette positive current of 5 mA, 2 s ON/2 s OFF pulses, 60–70 times) at three to four sites in lamina A of LGN. A group of kittens (n = 3) was deprived of vision in both eyes by eyelid suture. All surgical procedures were performed under sterile condition under anesthesia with 2–3% isoflurane in N2O and O2 (1:1). All the incisions were infiltrated with local anesthetics. The animals were administered an antimicrobial agent (enrofloxacin, 5 mg/kg) every day after the surgery until euthanasia. Retraction of Active Axons Histology After a survival period of one week, the cortical region inactivated by BoNT/E was delineated physiologically by mapping cortical cell activity with a tungsten microelectrode placed at various distances from the sites of BoNT/E injection. The kittens were anesthetized with pentobarbital (Nembutal, Dainippon Sumitomo Pharma; initial dose, 30 mg/kg; maintenance dose, 2–4 mg/kg/hr) during record- ing experiments. When no spike activity except for injury discharges was recorded while the electrode was advanced to a depth of 2000 mm, we determined the site to be inactivated by BoNT/E. A 27 G needle was inserted to form marks at the border of the inactivated region. Then the animals were euthanized with an overdose of pentobarbital (100 mg/kg, i.v.) and perfused transcar- dially with cold PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed and postfixed in the fixative containing 20% sucrose. Tissue blocks containing LGN and the entire caudal pole of the cortex were cut on a freezing microtome in the frontal plane (50 mm thickness). All sections were collected in PBS and processed for the standard ABC method. Briefly, the sections were incubated overnight at 4uC in a blocking solution containing 5% bovine serum albumin (BSA), 3% normal goat serum (NGS) and 0.7% Triton X-100 in PBS. They were then transferred to a solution containing the avidin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA), 0.1% Triton X-100 and 0.1% BSA in PBS and incubated overnight at 4uC. After washing in PBS and Tris-buffer (50 mM, pH 7.4), they were reacted with a solution containing 0.1% diaminobenzidine hydrochloride, 0.025% nickel ammonium sulfate, 0.025% cobalt chloride and 0.025% hydrogen peroxidase in Tris-buffer. Selected sections containing the visual cortex were processed for immunohistochemical staining of Egr-1. The sections were incubated with the blocking solution overnight at 4uC. They were then transferred to a solution of 2.5% BSA and 5% NGS in PBS containing an Egr-1 antibody at a dilution of 1:10000 (rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated overnight at 4uC. After washing in PBS and blocking, they were transferred to a solution containing 5% BSA, 2% NGS and 0.3% Triton X-100 in PBS containing a biotinylated secondary antibody (biotinylated goat anti-rabbit IgG; Vector Burlingame, CA, USA) and incubated for 1 hr at room tempera- ture. After washing in PBS, they were transferred to a solution containing avidin-horseradish peroxidase and incubated overnight at 4uC. After washing in PBS and Tris-buffer, they were reacted with a solution containing 0.01% diaminobenzidine hydrochloride, 0.025% nickel ammonium sulfate, 0.025% cobalt chloride and 0.035% hydrogen peroxidase in Tris-buffer. All sections were mounted on MAS-coated slides, dehydrated in a graded series of ethyl alcohol, cleared in xylene, and coverslipped. Selected sections containing LGN or the visual cortex were stained with cresyl violet for localization of layer boundaries and injection sites. Axonal arbor analysis All of the injection sites were well confined to a single lamina of LGN. When an injection site invades the other lamina representing the other eye, we eliminated all samples in the same animal from arbor reconstruction. We analyzed the axonal arbors in the area inactivated by BoNT/E defined physiologically. In addition, our analysis is limited to axons with well-filled arbors that could be followed up to their thinnest terminals. BDA-filled arbors were reconstructed at 61000 magnification from serial sections in 3D with the aid of a computer graphic system (Neurolucida, Microbrightfield, VT). We evaluated the size and complexity of each arbor by measuring the total length of the arbor and the number of branch points, respectively. The total length of each PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4193