Remodeling of synaptic structures in the motor cortex following spinal cord injury

Experimental Neurology 198 (2006) 401 – 415 www.elsevier.com/locate/yexnr

Remodeling of synaptic structures in the motor cortex following spinal cord injury Byung G. Kim a,1 , Hai-Ning Dai a , Marietta McAtee a , Stefano Vicini b , Barbara S. Bregman a,⁎ a

Department of Neuroscience, NRB Rm EP-04, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington, DC 20007, USA b Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007, USA Received 10 August 2005; revised 8 December 2005; accepted 9 December 2005 Available online 26 January 2006

Abstract After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI. © 2006 Elsevier Inc. All rights reserved. Keywords: Spinal cord injury; Dendritic spine; Synaptic remodeling; Motor cortex; PSD-95; PSA-NCAM

Introduction Spinal cord injury (SCI) removes supraspinal input to the spinal motor networks and thus results in a severe and permanent loss of motor function below the injury site. Some degree of functional recovery, however, can be observed without intervention (Bregman and Goldberger, 1983; Burns et al., 1997). Since regeneration of injured axons is extremely limited in mature CNS, spontaneous recovery in motor function appears to be mediated by structural reorganization of spared motor system. This compensatory remodeling occurs at

⁎ Corresponding author. Fax: +1 202 687 0617. E-mail address: [email protected] (B.S. Bregman). 1 Current address: Brain Disease Research Center, Ajou University School of Medicine, Suwon 443-721, Republic of Korea. 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.12.010

multiple levels of the neuraxis including spinal motor centers, descending supraspinal motor tracts, brainstem, and the motor cortex (Raineteau and Schwab, 2001). The sensorimotor cortex in adults retains the capability to reorganize in response to alteration in peripheral sensory input or behavioral manipulation (Clark et al., 1988; Nudo et al., 1996). A much greater extent of structural and functional plasticity can be observed after large-scale injuries such as limb amputation or SCI (Jain et al., 1997; Pons et al., 1991). Several lines of evidence suggest that synaptic connectivity in the sensorimotor cortex can be modified after SCI. Spinal lesions in cats and primates reshape the sensory representational map in the cortex (Jain et al., 1997; McKinley et al., 1987). Functional imaging (Bruehlmeier et al., 1998) and transcranial magnetic stimulation (Topka et al., 1991) demonstrated significant alterations in connectivity between the motor cortex and spinal cord in human SCI as well.

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The cellular and/or anatomical substrates subserving injuryinduced cortical plasticity are not fully understood. Unmasking or potentiation of existing connections may contribute to the plasticity within a short time scale (Hess and Donoghue, 1994; Jacobs and Donoghue, 1991), whereas alteration in cortical connectivity over months to years may involve new growth of axonal and dendritic processes (Darian-Smith and Gilbert, 1994; Florence et al., 1998; Volkmar and Greenough, 1972). Structural remodeling at the level of individual synapses may be another mechanism (Stepanyants et al., 2002). The density of dendritic spines, postsynaptic specializations at the excitatory synapses, is sensitive to a variety of experience or environmental stimuli (Globus et al., 1973; Gould et al., 1990; Moser et al., 1994). Recent in vivo imaging studies have suggested that the appearance and disappearance of spines underlie experience-dependent plasticity during development as well as in adults (Lendvai et al., 2000; Trachtenberg et al., 2002). Not only spine density but also morphology of spine structures is sensitive to various stimuli that are associated with synaptic plasticity (Hayashi and Majewska, 2005; Yuste and Bonhoeffer, 2001). For example, changes in spine neck length were observed after social stimulation or electrical stimulation to evoke long-term potentiation (Coss and Globus, 1978; Fifkova and Anderson, 1981), and changes in morphology or size of spine head were also associated with synaptic plasticity (Desmond and Levy, 1983, 1986; Matsuzaki et al., 2004). It is not known, however, whether remodeling of dendritic spines, particularly spine morphology, is involved in the injury-induced cortical reorganization, and if so, with what time scale this remodeling proceeds after injury. In this study, we sought to determine whether SCI leads to remodeling of synaptic structures in the motor cortex. For this end, we visualized dendritic spines in the motor cortex using confocal microscopy in fixed slice preparations and examined the spine density and morphology in detail. Since spine morphology such as spine length and head diameter is closely related to functional characteristics of an individual synapse (Kasai et al., 2003; Yuste et al., 2000), the analysis of spine morphology from a large population of dendritic spines also allowed us to infer the potential changes in overall functional properties in the motor cortical network following SCI. Furthermore, we examined correlative changes in expression of various synapse-associated proteins in the motor cortex after SCI, attempting to define a temporal profile of the remodeling process.

previously (Bregman et al., 1997). This injury removes the right hemicord plus the left dorsal column. We severed the dorsal corticospinal tract bilaterally because it is technically difficult to exclusively disrupt unilateral corticospinal tract. Rats were anesthetized with 4% chloral hydrate (400 mg/kg body weight). After a laminectomy, the dura was opened, and iridectomy scissors were used to create a spinal cord overhemisection. Vacuum suction was used to aspirate the remaining tissue at the right ventrolateral recess of the canal. Preparation of lightly fixed cortical slices

Materials and methods

For visualization of the dendritic spines, we chose to use confocal microscopic imaging because this method allows more sensitive detection of spines and more accurate measurement of morphology from a large number of samples. To label dendritic processes with the fluorescent dye DiI (1,1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate; catalogue # D-282; Molecular Probe, Eugene, OR), we used formaldehydefixed slices rather than live ones because a previous study reported that spine density rapidly increases during slice preparation (Kirov et al., 1999). At different time points after spinal lesion, rats were sacrificed and fixed by transcardial perfusion. Approximately 200 ml of heparinized saline was pumped into the left ventricle followed by 300 ml of 1.5% paraformaldehyde in 0.1 M phosphate buffer. In a pilot experiment, we found that filling and diffusion of DiI through the neuronal membrane are severely restricted in slices fixed with 4% paraformaldehyde. By lowering the concentration to 1.5%, we obtained a fine staining of neuronal processes comparable to that in live unfixed slices. This concentration of paraformaldehyde resulted in a better preservation of dendritic architecture compared to the live slice condition, where we observed frequent notching and swelling of the dendritic shafts, like a string-and-beads pattern, which is probably due to disassembly of cytoskeletal proteins (Fiala et al., 2003). After dissection, brains were postfixed in 1.5% paraformaldehyde for 1 h and then transferred to and maintained in cold phosphate-buffered saline (PBS) until being sliced. On the same day, brains were coronally sectioned into 200 μm slices with a vibratome (model Vibratome 1000 classic; Technical Products Int'l. Inc., O'Fallon, MO). Slices containing the forelimb motor cortex were collected in PBS. The rostrocaudal extent of the forelimb motor cortex was determined according to the previous mapping study (Donoghue and Wise, 1982). Typically, we took slices located between +1.7 mm and +0.4 mm from bregma.

Animals and spinal lesion

DiI labeling procedure

Adult female Sprague–Dawley rats (200–250 gm; Zivic Inc., Zelienople, PA) were used in this study. They were housed in the Georgetown University Division of Comparative Medicine Facility and all protocols were approved by the Georgetown University Animal Care and Use Committee. Animals received a right cervical overhemisection injury at the C4 level using a procedure modified from that described

Solid DiI crystals were applied onto the slices under a dissecting microscope. Rather than using a gene-gun mediated delivery in “DiOlistic” labeling (Gan et al., 2000), we used a glass micropipette, which was specially polished to have a sharp and elongated tip, to deliver very fine crystals on cortical slices. By lightly touching or sometimes gently poking the slices with a pipette tip covered with DiI crystals, we were able to get a fine

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Golgi-like staining with the dendritic arborization visualized in detail (Figs. 1A, B). DiI crystals occasionally clumped together, but stained dendritic processes outside of the clumps were usually well separated from each other and readily visualized. This method allowed us to control the layer to which DiI crystals are applied because the cortical layers are easily identified with illumination under a dissecting microscope. Therefore, we delivered DiI crystals separately onto superficial (layer II/III) and deep layer (V/VI) in the motor cortex. The lateral border of the forelimb motor cortex could be identified by a transition of luminance pattern from the granule cell layer in the S1 area to the agranular cortex under illumination. After labeling, slices were incubated in PBS at room temperature for 4 to 12 h to allow DiI crystals to diffuse fully along the neuronal membranes. Slices were then fixed again with 4% paraformaldehyde for 30 min and then washed in PBS three times each for 5 min. To avoid possible dehydration-induced shrinkage of dendritic structures (Trommald et al., 1995), we used glycerolbased mounting medium Mowiol (containing DABCO as an antifade reagent; Calbiochem, La Jolla, CA) instead of xylene. All images were taken within 7 days after coverslipping. Before image capture, all slides were assigned randomized codes to ensure a blind evaluation of the experimental conditions.

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Laser confocal microscopic imaging of dendritic segments Spiny pyramidal neurons were analyzed in this study. We imaged dendritic spines using a Zeiss 510 Meta confocal laser scanning microscope (LSM 510 META). Dendritic segments that were well separated from neighboring neural processes were randomly sampled in both superficial and deep layers of the forelimb motor cortex. For basal dendrites, dendritic segments that run tangentially or towards the white matter were imaged. Each dendritic segment we imaged belonged to a different neuron. All images were taken using the PlanAPOCHROMAT 63× oil-immersion lens (N/A 1.4). A 543 nm Helium/Neon laser was used to visualize fluorescence emitted by DiI. The configuration parameters were as follows: (1) filters, channel 3 band pass 560–615 nm; (2) pinhole diameter, 108 μm; (3) beam splitters, MBS-HFT UV/488/543, DBS1 mirror, DBS2 NFT, DBS3-plate. Since the original signal intensity differed greatly from image to image (due to a variability in staining quality, level of background, and the extent of bleaching), we had to use different detector settings (especially detector gain) to maximize dynamic range for each image. After acquisition, brightness and contrast were adjusted to have similar signal intensity in the parent dendrites. We used

Fig. 1. Visualization of dendritic spines in fixed slice preparation. (A) DiI crystals applied with a glass micropipette on lightly fixed cortical slices were used to stain neuronal processes. DiI crystals occasionally clumped together, but stained dendritic processes outside of the clumps were readily visible. Scale bar = 100 μm. (B) At higher magnification, fine dendritic architecture was visualized, resembling Golgi-stained preparations. For confocal microscopic imaging of the basal dendrites, we took dendritic segments that run tangentially or towards the white matter and are well separated from neighboring neural processes. Scale bar = 50 μm. (C) Examples of confocal microscopic images with the parameters described in Materials and methods; left panel, dendritic segment with an axon running behind it in the superficial (2/ 3) layer; right panel, a basal dendrite in the deep (5/6) layer. The spine density is generally lower in the deep than in the superficial layer. Scale bar = 5 μm. (D) High magnification image from the boxed region in panel C. This magnification was typically used for quantification of spine morphology as illustrated by the lines with arrows. L = spine length, D = head diameter.

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2048 × 2048 pixels for frame size without zooming. Serial stack images with step size ranging from 0.4 to 0.6 μm were collected, and then projected to reconstruct a three-dimensional (3D) image. Image analysis Before image analysis, we tried to adjust the brightness of all images using the Zeiss LSM Image Browser software (version 3.2) to the level where dendritic signal is almost fully saturated (see Fig. 1C). Both a series of stack images and a 3D projection image were used in a complimentary manner to increase the sensitivity of spine detection. Dendritic spines were identified and counted in the 3D projection image interface. Whenever dendritic spines were too crowded to separate them from each other, we turned to serial stack images to delineate individual spines (Trommald et al., 1995). By scrolling through the stack of different optical sections, individual spine heads could be identified with greater certainty. All dendritic protrusions with a clearly recognizable neck were counted as spines. When a protrusion was connected to a parent dendrite without a clear neck or stalk, it was counted as a spine only when there was a clear indentation at the either side of junction of the protrusion and dendrite to differentiate a spine from a dendritic kink. Spine number was divided by the length of dendritic segment (average length of the segments analyzed from all groups, 28.1 ± 0.40 μm, see Tables 1 and 2) to generate dendritic spine density expressed as number/μm. Head diameter was defined as the length of longest straight line through the spine head orthogonal to the neck of spine (Fig. 1D). Spine length was defined as the distance from the outer edge of the dendritic shaft and to the tip of the spine head traced by a polyline drawing tool in the Zeiss Image Browser software (Fig. 1D). For basal dendrites, we took dendritic segments at least 30 μm away from the cell body, because we found that spine density is consistently lower at the dendritic segment very close to the cell body. Dendritic segments were not selected on the basis of branching order Table 1 Summary of the measured variables in the superficial layer

Number of animals Number of dendritic segments Average/total length of dendrites analyzed (μm) Number of spines Spine density (number/μm) Mean Median Coefficient of variation Head diameter (μm) Mean Median Coefficient of variation Spine length (μm) Mean Median Coefficient of variation

CTR

7D

28D

5 34 28.8/980.5

5 42 26.2/1098.8

4 30 27.5/825.8

1866

1834

1437

1.89 1.87 13.5%

1.69 1.65 14.3%

1.75 1.76 13.8%

0.571 0.55 27.9%

0.584 0.56 28.3%

0.570 0.55 27.0%

1.90 1.79 39.2%

1.99 1.88 39.4%

2.09 1.97 43.9%

CTR = control, 7D and 28D = 7 and 28 days after spinal cord injury.

Table 2 Summary of the measured variables in the deep layer

Number of animals Number of dendritic segments Average/total length of dendrites analyzed (μm) Number of spines Spine density (number/μm) Mean Median Coefficient of variation Head diameter (μm) Mean Median Coefficient of variation Spine length (μm) Mean Median Coefficient of variation

CTR

7D

28D

5 31 28.3/878.2

5 42 29.0/1217.4

4 16 29.5/471.5

1346

1639

696

1.55 1.53 18.2%

1.35 1.30 18.3%

1.46 1.38 18.9%

0.584 0.56 26.6%

0.585 0.56 27.5%

0.615 0.60 27.2%

1.95 1.83 41.1%

2.01 1.86 41.3%

2.18 1.99 43.4%

CTR = control, 7D and 28D = 7 and 28 days after spinal cord injury.

because the pattern of dendritic branching was highly variable between different neurons, and it was impossible to define an exact order in some dendritic segments due to the branches growing in a Z-direction. For apical dendrites, DiI staining with our method usually did not visualize the entire length from the cell body to the pial tip, so we could not determine the distance from the cell body in many occasions. Therefore, we only analyzed the data obtained from the basal dendrites. Immunoblot analysis A separate subset of animals were briefly perfused with icecold saline to remove blood cells at different time points after SCI. Tissue samples were homogenized with a dounce tissue grinder in ice-cold homogenization buffer (50 mM Tris–HCl, pH 7.6, containing 150 mM NaCl, 1 mM EDTA, 0.32 M sucrose, 100 mM sodium vanadate) supplemented with 1× Complete protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentration of the homogenates was measured using Micro BCA Protein Assay kit (Pierce, Rockford, IL), and equal amounts of proteins (typically 10 to 20 μg) were resolved on a 4–12% gradient or 8% gel and transferred to a PVDF membrane. The membrane was blocked in 2.0% BSA in Tris-buffered saline (TBS) and probed with primary antibody diluted in TBS containing 0.2% Tween-20 for 2 h at room temperature or overnight at 4°C. Primary antibodies used in this study were mouse anti-synaptophysin (1:1000; Sigma, St. Louis, MO), mouse anti-PDS-95 (1:000; Affinity Bioreagents, Golden, CO), mouse anti-NR1, mouse anti-NR2B, rabbit antiNR2A (1 μg/ml; generous gifts from Dr. Barry Wolfe, Georgetown University, Washington, DC), rabbit anti-GABAA receptor α1 (1:2000; Affinity Bioreagents, Golden, CO), rabbit anti-GAD 65/67 (1:2000; Chemicon, Temecula, CA), mouse anti-PSA-NCAM (1:500; Chemicon, Temecula, CA), and rabbit anti-NCAM (1:5000; Chemicon, Temecula, CA). When needed, blots were stripped with Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) for 5 to 15 min at 37°C

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and reprobed with the second primary antibody. After incubation with peroxidase-conjugated anti-mouse IgG (1:50,000; Pierce, Rockford, IL) or anti-rabbit IgG (1:5000; Abcam, Cambridge, MA), immunoreactivity was detected with Super Signal West Pico (Pierce, Rockford, IL). All blots were also probed with rabbit anti-β actin or rabbit anti-GAPDH (1:5000 for both; Abcam, Cambridge, MA) to control differences in loading amount. Optical density of band signals was quantified with the publicly available software Image J (http://rsb.info.nih.gov/ij/) and normalized by relative beta actin or GAPDH signals on the same blot. Each value in a blot was expressed as a percent of controls run in the same blot.

followed by post hoc Tukey's multiple comparison test. Distribution of both spine head diameter and spine length did not conform to the Gaussian, normal distribution (P b 0.0001 by one-sample Kolmogorov–Smirnov test; for both head diameter and spine length). Therefore, nonparametric Kruskal–Wallis test was used to compare group means for spine head diameter and length, followed by Mann–Whitney U test for post hoc comparison between each group. Statistical analysis was performed with SPSS version 12.0 (Chicago, IL) or GraphPad Prism software version 4.0 (San Diego, CA).

Statistical analysis

Remodeling of dendritic spines in the motor cortex following SCI

Two-sample Kolmogorov–Smirnov test was used to compare the patterns of cumulative or relative frequency plots in the histogram of individual values for spine density, head diameter, and length. For comparison of mean values between groups, we treated each dendritic segment (equivalent to each neuron in this study) for density analysis or each spine for morphology analysis as an independent observation. Comparison of group means in spine density was performed with one-way ANOVA

Results

To determine whether axotomy at the spinal level alters synaptic structures in the motor cortex, we focused on postsynaptic spine structures and measured density (number per unit length), head diameter, and length of dendritic spines. To more accurately quantify those variables from a large number of spines, we opted to use confocal microscopic imaging on fluorescently labeled neuronal processes, instead of

Fig. 2. Density of dendritic spines in the motor cortex following spinal cord injury. (A, B) Spine density from basal dendrites in the superficial layer. (C, D) Spine density from basal dendrites in the deep layer. (A) Cumulative frequency histogram of spine density in the superficial layer. (B) Comparison of mean spine density from the same set of data as in panel A. (C) Spine density distribution in the cumulative frequency histogram in the deep layer. (D) Comparison of mean spine density from the same set of data as in panel C. Number in parenthesis indicates the number of dendritic segments analyzed in each group. The error bars indicate SEM. Asterisks (**) indicate significant difference at P b 0.01 compared to control (CTR) by Mann–Whitney U test.

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using traditional Golgi staining or electronmicroscopy (EM) analysis. Golgi staining tends to substantially underestimate spine density due to the geometric obscureness by the opacity of the Golgi-impregnated dendritic shafts (Feldman and Peters, 1979). Confocal microscopic imaging produces a series of stack images covering three-dimensional depths of spines and dendrites, allowing a more sensitive detection of spines by scrolling forward and backward through the entire series of stack images. Although the highest resolution in spine imaging can be achieved with EM, time and labor-intensive nature of EM procedures would hardly allow a large sample size adequate for proper representation of the population of interest. The resolution of confocal images obtained with our parameters (see Materials and methods) allowed us accurate quantification of head diameter and spine length (Fig. 1D). Details of measured variables from the superficial and deep layer are summarized in Tables 1 and 2, respectively. Spine density We obtained the density of dendritic spines in the left forelimb motor cortex at 7 and 28 days (7D and 28D group, respectively) after a right cervical overhemisection injury at the C4 segment to examine possible changes at early and later time

point, respectively. More than 90% of corticospinal axons originating from the left motor cortex are expected to be severed by this lesion paradigm. This injury results in a severe and permanent impairment of right forelimb function (Bregman et al., 2002). Control (CTR) rats received a laminectomy without a cord injury and were sacrificed 7 days after the sham operation. The number of animals used in spine analysis was 5 for CTR, 5 for 7D, and 4 for 28D group (see Tables 1 and 2). Spine density of the basal dendrites in the superficial layer in CTR rats ranged between 1.35/μm and 2.52/μm (Fig. 2A). Axotomy led to a decrease in spine density by 7 days after injury. Lesioning the spinal cord at the C4 segment resulted in a left shift of spine density distribution in the cumulative frequency histogram from the superficial layer in 7D group, indicating a decrease in spine density at 7 days after axotomy. The Kolmogorov–Smirnov test revealed a significant difference in the distribution of spine density between CTR and 7D groups (P = 0.007). The cumulative frequency plot from 28D group also shifted to the left compared to control group, but the difference in distribution was not significant (P = 0.207). Furthermore, there was a 10.3% decrease in the mean spine density of the basal dendrites in the superficial layer in 7D group compared to that of CTR (Fig. 2B). Mean spine density in 28D group was still lower compared

Fig. 3. Changes in spine head diameter after spinal cord injury. (A, B) Head diameter from the spines in the superficial layer. (C, D) Head diameter in the deep layer. (A) Frequency histogram of spine head diameter in the superficial layer. (B) Comparison of mean spine head diameter from the same set of data as in panel A. (C) Relative frequency plots of head diameter in the deep layer. (D) Comparison of mean head diameter from the same set of data as in panel C. Number in parenthesis indicates the number of dendritic spines analyzed in each group. The error bars indicate SEM. Asterisks indicate significant difference at P b 0.05 (*) and P b 0.01 (**) compared to control (CTR) by Mann–Whitney U test.

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to CTR, but was higher than that of 7D. A significant group difference was revealed by one-way ANOVA (F(2,103) = 6.149, P = 0.003), and Tukey's post hoc analysis showed a significant difference only between CTR and 7D groups (P b 0.01). Spine density was generally lower in the deep layer basal dendrites than that in the superficial layer (Fig. 1C), so the range was from

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0.87/μm to 2.18/μm in CTR group (Fig. 2C). A very similar pattern to the superficial layer was observed in the cumulative frequency histogram of spine density from the deep layer basal dendrites. A cumulative frequency plot significantly shifted to the left in 7D group (Kolmogorov–Smirnov test, P = 0.015), and again the density distribution from 28D group was

Fig. 4. Changes in the length of dendritic spines after spinal cord injury. (A, B) Spine length from the spines in the superficial layer. (C, D) Spine length in the deep layer. (A) Frequency histogram of spine length in the superficial layer. (B) Comparison of mean spine length from the same set of data as in panel A. (C) Frequency histogram of spine length in the deep layer. (D) Comparison of mean spine length from the same set of data as in panel C. Number in parenthesis indicates the number of dendritic spines analyzed in each group. The error bars indicate SEM. Asterisks (**) indicate significant difference at P b 0.01 compared to control (CTR) by Mann– Whitney U test. (E) A representative example of superficial basal dendrites in the 28D group shows an increased proportion of longer spines compared to CTR and 7D groups. Scale bar = 5 μm.

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positioned between those of CTR and 7D groups (CTR vs. 28D, P = 0.406). Comparison of the mean spine density showed a significant decrease only in 7D group (by 12.9%; one-way ANOVA, F(2,86) = 5.143, P = 0.0078; Tukey's post hoc, P b 0.01) (Fig. 2D). Taken together, the analysis of dendritic spines in the forelimb motor cortex suggests that roughly 10% of dendritic spines are lost by 7 days after a cervical spinal axotomy. The initial loss seems to be followed by a partial recovery by 28 day after SCI. This change occurred not only in the deep layer but also in the superficial layer in which the neurons are not directly injured by SCI. Spine head diameter A total of 8648 spines (5137 from the superficial and 3681 from the deep layer basal dendrites, same set of dendrites for spine density analysis) were included in the analysis of head diameter and spine length (Tables 1 and 2). In the superficial layer, although there was no significant difference in the distribution pattern of head diameter in the frequency histogram (Kolmogorov–Smirnov test, CTR vs. 7D, P = 0.14; CTR vs. 28D, P = 0.845) (Fig. 3A), we found a small, but statistically significant effect of spinal injury on mean head diameter (Kruskal–Wallis test, P = 0.034) (Fig. 3B). Post hoc Mann–Whitney U test revealed a significant increase only in 7D group (by 2.3%, CTR vs. 7D, P = 0.017; CTR vs. 28D, P = 0.825). Interestingly, the head diameter of dendritic spines from the deep layer basal dendrites showed a different time course of change. There was a significant shift towards the right of head diameter distribution in the frequency histogram only in 28D group (Kolmogorov–Smirnov test, CTR vs. 7D, P = 0.996; CTR vs. 28D, P b 0.001) (Fig. 3C). Comparison of mean head diameter also showed a significant increase in 28D group by 5.4% (Kruskal–Wallis test, P b 0.001; Mann– Whitney U test, CTR vs. 28D, P b 0.001) (Fig. 3D). Thus, spinal injury seems to increase spine head diameter with a different temporal course depending on the layer to which analyzed dendrites belong: the increase occurred in the superficial layer at 7 days and in the deep layer at 28 days after SCI.

Spine length In the superficial layer, the frequency histogram of spine length showed an apparent right shift of a relative frequency plot in 28D group, suggesting that SCI increased the proportion of longer spines by 28 days after injury (Fig. 4A). Spine length distribution in 7D group was also shifted to the right but to a smaller extent. There were significant differences in spine length distribution in the frequency histogram in both 7D and 28D groups compared to CTR (Kolmogorov–Smirnov test, CTR vs. 7D, P = 0.002; CTR vs. 28D, P b 0.001). Mean length of dendritic spines increased by 4.5% and 9.7% at 7 and 28 days after SCI, respectively (Fig. 4B). Kruskal–Wallis test showed a significant group effect (P b 0.001) and post hoc Mann– Whitney U test revealed significant differences in both 7D and 28D groups compared to CTR (CTR vs. 7D, P = 0.001; CTR vs. 28D, P b 0.001). Spine length in the deep layer showed a similar pattern to that in superficial layer. The proportion of longer spines was apparently increased at 28 days after SCI, whereas a relative frequency plot of spine length in 7D group was not significantly different from that in CTR group (Kolmogorov– Smirnov test, CTR vs. 7D, P = 0.081; CTR vs. 28D, P b 0.001) (Fig. 4C). Comparison of mean spine length from the deep basal dendrites revealed a significant group effect by spinal injury (Kruskal–Wallis test, P b 0.001), and post hoc Mann–Whitney U test revealed a significant increase in 28D group (by 12.0%, P b 0.001), although the increase in 7D did not reach statistical significance (by 3.2%, P = 0.071). These results indicate that axotomy at the spinal level leads to an increase in the length of dendritic spines in the motor cortex. In some dendritic segments, the increase in elongated spines was visibly apparent (Fig. 4E). The spine lengthening was already detectable at 7 days, but became more apparent at 28 days after injury, when the amount of change reached almost 10%. Spine lengthening occurred similarly between the superficial and deep layers, suggesting that the spine length is globally regulated in the whole motor cortical network rather than in a layer-specific manner. There is substantial evidence that dendritic protrusions are longer during early development and become shorter as mature

Fig. 5. Filopodium-like long dendritic protrusions. (A) Examples of filopodium-like long protrusions (arrows) defined as longer than 5 μm in length. Left panel, a superficial layer dendrite at 7 days. Right panel, a deep layer dendrite at 28 days. Scale bar = 2 μm. (B) Percentage of filopodium-like protrusions out of total dendritic spines analyzed at each time point. Frequency of filopodium-like protrusion is increased at both 7 and 28 days after SCI. Asterisks indicate significant differences at P b 0.05 (*) and P b 0.01 (**) compared to control (CTR) by Chi-square test.

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synaptic connections are established (Nimchinsky et al., 2001; Portera-Cailliau et al., 2003; Ziv and Smith, 1996). Lengthening of dendritic spines may thus indicate that the property of synaptic connections in the motor cortex is rendered immature by SCI. Another feature of immature synaptic connectivity is the presence of filopodium-like long dendritic protrusions, which are exclusively observed during development (Miller and Peters, 1981; Purpura, 1975). Filopodial dendritic protrusions are thought to make initial contacts with presynaptic partners and thus be involved in early synaptogenesis (Fiala et al., 1998; Ziv and Smith, 1996). During the course of spine analysis, we were able to observe long filopodium-like dendritic protrusions (Fig. 5A), although they were very rarely found. We analyzed observation frequency of the filopodium-like dendritic protru-

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sions defined as longer than 5 μm in length. Because the absolute number of such long protrusions was too small, the numbers from both layers were lumped together in this analysis. As expected, only 0.16% spines (5 out of 3212 spines from both superficial and deep layers) were more than 5 μm in CTR group (Fig. 5B). The frequency of filopodium-like protrusions went up 2.7-fold (0.43%) by 7 days after SCI, and even further increased by 28 days to 0.89%, which was an almost five-fold increase compared to CTR. The difference in observation frequency was statistically significant at both 7 and 28 days after SCI by Chisquare test (CTR vs. 7D, χ2 = 4.269, P = 0.0388; CTR vs. 28D, χ2 = 13.89, P = 0.0002). The higher observation frequency of filopodium-like protrusions may suggest an increase in the incidence of synaptogenic events in the motor cortex after SCI.

Fig. 6. Expression of synaptic proteins in the motor cortex following spinal cord injury (SCI). Motor cortex tissues obtained 0 (control, CTR), 3, 7, 14, and 28 days after SCI were homogenized and subjected to immunoblot analysis. Optical density from each band was normalized by the relative beta-actin level probed in the same blot, and the data were expressed as % of control run in the same blot. (A) A representative blot and quantification graph for synaptophysin. N = 6 per each group. (B, left panel) Expression of PSD-95, a major postsynaptic density protein, is increased in the motor cortex at 14 days after SCI (N = 7 to 8 per each group). Right panel, PSD-95 expression in the visual cortex (VC) in CTR and 14D groups. Asterisk (*) indicates significant difference at P b 0.05 compared to CTR by ANOVA followed by Tukey's post hoc analysis. The error bars indicate SEM. Approximate molecular weight is indicated in kiloDalton (KD) unit.

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Immunoblot analysis of synapse-associated proteins The analysis of dendritic spines suggested that a dynamic process of synaptic structural remodeling goes on in the motor cortex during a 4-week period following SCI. In order to get an insight into possible molecular mechanisms associated with the process, and, more importantly, to determine a more detailed temporal profile of the synaptic remodeling process, we examined the expression of various synapse-associated proteins in the motor cortex 3, 7, 14, and 28 days after SCI (3D, 7D, 14D, and 28D, respectively).

Despite the observed changes in postsynaptic spine structures, expression of the presynaptic marker synaptophysin, which has been used as a marker of synaptogenesis (Masliah et al., 1991), did not change significantly at any time point examined (F(4,25) = 0.1879, P = 0.9425) (Fig. 6A). Expression of PSD-95, a major postsynaptic density protein, increased slightly, but significantly only at 14D after SCI (130.3 ± 8.7%; F(4,33) = 4.517, P = 0.0053; Tukey's post hoc, P b 0.05) (Fig. 6B). This change seems to be directly associated with a spinal lesion because PSD-95 level did not change in the visual cortex (100.9 ± 8.9%, Fig. 6B, right

Fig. 7. Expression of polysialylated neural cell adhesion molecule (PSA-NCAM) in the motor cortex after spinal cord injury (SCI). (A) A representative blot and quantification graph for PSA-NCAM. N = 7 to 8 per each group. (B) The same blot in panel A was stripped and reprobed with anti-NCAM antibody. Two bands each representing 180 and 140 kDa isoforms are visualized. A third band at 120 kDa range is also faintly visible. Quantification plots for 180 and 140 kDa bands are shown separately. N = 7 to 8 per each group. Asterisk (*) indicates significant difference at P b 0.05 compared to control by ANOVA followed by Tukey's post hoc analysis. The error bars indicate SEM. Approximate molecular weight is indicated in kiloDalton (KD) unit.

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panel) which does not have any axonal connection with the spinal cord. Since NMDA receptors are physically associated with PSD-95 at the postsynaptic density, we also examined the expression of NMDA subunit proteins. NR1 showed a tendency towards increase also at 14D (122.6 ± 7.8%), but it was not statistically significant (F(4,33) = 2.190, P = 0.0917). NR2A and NR2B proteins also did not show any significant alteration (data not shown). In addition, proteins involved in inhibitory synaptic transmission such as GABA-A receptor α1 subunit and GABA synthesizing enzyme GABA 65/67, did not change significantly (data not shown). The lack of changes in the proteins involved in the NMDA and GABA systems may suggest that quantitative expression of those proteins is not globally regulated in the motor cortex after SCI. Pre- and postsynaptic membranes are not simply apposed to each other at the synaptic junction, but they are tightly connected by various adhesion molecules (Benson et al., 2000). One of them, neural cell adhesion molecule (NCAM), is expressed at synapses particularly the postsynaptic density, and thus thought to be critical in determining the stability of synaptic structure (Persohn et al., 1989). Its polysialylated form, PSA-NCAM, is considered being less adhesive and thought to represent active synaptic remodeling (Rutishauser and Landmesser, 1996). It is likely that the removal and/or formation of dendritic spines are preceded or at least accompanied by attenuation of adhesiveness between the pre- and postsynaptic structures. We determined whether the expression level of PSANCAM in the motor cortex is correlated with spine remodeling after SCI. PSA-NCAM level started to increase as early as 3D and remained elevated until the 14D time point when it reached a peak level (3D = 145.5 ± 14.8%, 7D = 150.3 ± 12.0%, 14D = 159.3 ± 15.6%; Fig. 7A). The expression abruptly went down close to control value at 28D. One-way ANOVA revealed a significant group effect among different time points examined (F(4,33) = 4.968, P = 0.002). Tukey's post hoc showed significant differences only at 14D (3D, P = 0.087; 7D, P = 0.058; 14D, P = 0.013), probably due to a higher variability at the earlier time points. Again, the increase was not detected in the visual cortex (97.3% at 14D). The same blots for PSANCAM were stripped and reprobed with anti-NCAM antibody. In our experimental condition, two isoforms with 180 kDa and 140 kDa each were readily visualized. NCAM-180, to which PSA is mainly linked (6), showed a tendency to increase at 3D and 14D (3D = 123.7 ± 7.9%, 14D = 138.4 ± 15.8%), although the amount of increase was less than that of PSA-NCAM (Fig. 7B). A significant group effect was revealed by one-way ANOVA (F(4,33) = 2.984, P = 0.033), but Tukey's post hoc did not show any significance difference at all time points (3D, P = 0.479; 14D, P = 0.076). NCAM-140 also did not show any significant alteration, indicating that there is a selective upregulation of PSA-NCAM rather than an overall increase in all NCAM species. Taken together, these results suggest that regulation of PSA-NCAM expression may be involved in remodeling of synaptic structures in the motor cortex after SCI. In addition, the temporal profile of PSA-NCAM upregulation (from 3 to 14 days) may provide a clue in discerning the

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temporal window of synaptic remodeling process that takes place in the motor cortex after SCI. Discussion We made several observations that collectively provide evidence that dynamic remodeling of synaptic structures occurs in the motor cortex following SCI. First, the density of postsynaptic spines decreases in the motor cortex at 7 days after SCI followed by a partial recovery by 28 days. Second, spine head diameter increases after SCI with a different time course depending on the layer. Third, SCI leads to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean length. The spine lengthening was accompanied by a higher frequency of filopodium-like long dendritic protrusions. Fourth, PSD-95, a major postsynaptic density protein, and PSA-NCAM, a marker of active synaptic remodeling, increase their expression at 14 days after SCI. These results further suggest that synaptic remodeling may involve both synapse elimination and formation of new synapses with a distinctive time course, and that the remodeling process proceeds with a time scale ranging from days to weeks after injury. Dendritic spine loss following deafferentation has been reported in several models of CNS injury (Cheng et al., 1997; Parnavelas et al., 1974; Schauwecker and McNeill, 1996). Unlike these models where axonal inputs to the spines are directly disrupted, however, anatomical integrity of afferents to the motor cortex is presumably preserved after SCI. Instead, efferent connections from the motor cortex to the spinal motor centers are directly severed by the lesion, resulting in an impairment of forelimb usage in our model (Bregman et al., 2002). The profound deficit in forelimb usage may be accompanied by depressed synaptic or neuronal activity in the forelimb motor cortex. Spine density in cultured organotypic slices is closely related to the level of activity (Annis et al., 1994; McKinney et al., 1999). A decrease in the activity of motor cortical circuitry may thus cause a transient spine loss. Altered synaptic connectivity develops in the motor cortex after SCI as the injured and/or spared corticospinal axons reorganize to get wired to new targets (Bareyre et al., 2004; Weidner et al., 2001). For example, microstimulation of the hindlimb motor cortex after a thoracic spinal lesion evokes motor responses in different body parts such as the forelimb, whisker, and trunk (Fouad et al., 2001), suggesting that the original connectivity has been replaced with new one. The transient decrease in spine density at 7 days may be related to a process that eliminates synaptic connections that are no longer used. A reduction in spine density was no longer significant at 28 days after SCI, suggesting a partial recovery of spine density by that time point. The potential synaptic elimination process during early days after SCI, therefore, might be followed by formation of new synaptic connectivity. The most prominent change in spine morphology was an increase in spine length at 28 days after SCI. It has been postulated that the length of dendritic spines is developmentally regulated (Portera-Cailliau et al., 2003). For example, mean

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spine length decreases by 17% between 1 and 4 weeks in the mouse barrel cortex (Nimchinsky et al., 2001). The amount of increase in spine length we observed after SCI was 10%, which seems to be a remarkable extent as compared to the developmental change. The appearance of shorter spines coincides with increasing developmental age (Dailey and Smith, 1996), suggesting that longer spines in general are more immature than shorter counterparts. The increase in spine length after SCI may thus indicate that functional status of synaptic connectivity is changed into a more immature one. The spine lengthening may be a consequence of remodeling process in synaptic structures. Having eliminated obsolete connections, the motor cortex might generate new synaptic connections accompanied by spine formation. Newly generated spines are longer than existing ones, leading to an increased proportion of longer spines. In this context, it is quite intriguing that filopodium-like long protrusions are much more frequently observed after SCI. Dendritic filopodia are precursors of mature spines and involved in an early process of synaptogenesis (Dailey and Smith, 1996; Fiala et al., 1998; Ziv and Smith, 1996). Chronic blockade of synaptic transmission, which might mimic a decrease in synaptic activity in the forelimb motor cortex after the SCI in this model, was accompanied by an increase in dendritic filopodia (Petrak et al., 2005). An increase in filopodium-like protrusions may mirror a higher incidence of synaptogenic events in the motor cortex that is being reorganized after SCI. Given the close relationship between spine morphology and function (Hayashi and Majewska, 2005; Kasai et al., 2003; Yuste et al., 2000), it is highly likely that the changes in spine morphology, especially in spine length, are accompanied by significant functional consequences. It was experimentally demonstrated that spine neck offers a significant barrier to diffusion of calcium ions that are the most important mediator of synaptic plasticity (Svoboda et al., 1996), and the length of spine neck or spine neck geometry determines synaptic calcium dynamics (Majewska et al., 2000; Noguchi et al., 2005). Therefore, increased proportion of longer spines after SCI may result in an overall decrease in the rate of calcium diffusion through the neck by raising resistance from spine head to dendrite (Majewska et al., 2000), which may in turn lead to a larger and/or longer accumulation of calcium ions in spine heads in response to synaptic stimulation. Thus, synaptic connectivity in the motor cortical network may become more modifiable after SCI. In fact, longer spines are considered more vulnerable to morphological plasticity (Parnass et al., 2000). It is also possible that the lengthening of dendritic spines in adult cortex indicates an altered synaptic function. Elongated spines have been classically described in human mental retardation (Purpura, 1975). In addition, mean spine length was significantly longer in a mouse model of fragile X syndrome, the most common form of inherited mental retardation (Comery et al., 1997; Nimchinsky et al., 2001). These studies suggest a possibility that the SCI-induced spine lengthening in the forelimb motor cortex may be somehow related to the deficit in forelimb motor function in this injury model (Bregman et al., 2002).

We also observed a small, but statistically significant increase in spine head diameter after SCI. Spine head size is proportional to a release probability of presynaptic vesicles (Murthy et al., 2001; Schikorski and Stevens, 1997), and also to the amount of AMPA-type glutamate receptors (Matsuzaki et al., 2001; Takumi et al., 1999), suggesting that spines with bigger heads have stronger synapses. The increase in head diameter, in association with a decrease in spine density, may reflect a homeostatic mechanism to maintain the overall synaptic strength in a given network (Murthy et al., 2001; Turrigiano and Nelson, 2004; Wallace and Bear, 2004). Unlike the alteration in spine length where the similar pattern of change was observed in both superficial and deep layers, head diameter was differentially regulated between the two layers. Interestingly, the head diameter increase occurs earlier (at 7days after SCI) in the superficial than deep layer, although the corticospinal neurons that are directly affected by a spinal lesion reside in the deep rather than superficial layer. Reorganization of intracortical horizontal connections in the superficial layer after SCI may thus be regulated differentially from synaptic connections of the deeper layer. Since the changes in spine length and density were not different between the layers, however, it remains to be determined whether such a layerspecific mechanism is indeed in operation in this model. The results in this study demonstrate, for the first time, that SCI leads to time-dependent changes in density and morphology of dendritic spines in the motor cortex, suggesting that the remodeling of dendritic spines underlies cortical reorganization following SCI. Previous studies with unilateral cortical injury model have reported growth of dendritic processes in the contralateral cortex (Jones and Schallert, 1992, 1994). This dendritic overgrowth was paralleled by an increase in the number of synapses per neuron measured by electronmicroscopy at 30 days after injury (Jones et al., 1996), suggesting that synaptic remodeling is also involved in injury-induced reorganization in the contralateral cortex. Similarly, increased spine density measured by Golgi-staining was observed following callosal transection (Adkins et al., 2002). Spine density in the somatosensory cortex contralateral to an infarct, however, did not seem to increase in the only study that used confocal microscopy (Johansson and Belichenko, 2002). None of the above studies systematically analyzed changes in spine morphology. Our data that spine head diameter and length are significantly changed after SCI strongly suggest that alteration in spine morphology is implicated in injury-induced cortical reorganization. Images acquired from fixed slices allow only static description of spine structures at specified time points. A serial time-lapse in vivo imaging study revealed that about 50% of the spine population appears and disappears while the overall density is kept constant (Trachtenberg et al., 2002). It is possible that this rate of constitutive spine remodeling is greatly increased by a spinal lesion. In support of this notion, we found increased spine length and higher incidence of filopodium-like protrusions even at 7 days when the spine density drops below control level. This may indicate that the spine loss is accompanied by a certain extent of new spine formation at 7 days, but the balance tips towards a net loss by roughly 10%.

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Further increase in spine length along with more frequent occurrence of filopodium-like protrusions at 28 days may suggest that the rate of spine remodeling becomes even higher between these two time points and the balance tips towards formation of new spines. Immunoblot analysis of synapse-associated proteins provides supporting evidence for this argument. PSD-95 is implicated in synaptogenesis because it is promptly mobilized to new postsynaptic sites (Friedman et al., 2000). In addition, it has been used as a marker of postsynaptic remodeling (Marrs et al., 2001; Okabe et al., 1999). Upregulation of PSD-95 at 14 days after SCI may indicate a larger extent of postsynaptic remodeling with more frequent synaptogenic events between 7 and 28 days. More definitive evidence was that PSA-NCAM increases in the motor cortex at the same 14 days. Creating steric hindrance through its large hydrated volume or extensive negative charge, addition of PSA to NCAM decreases not only NCAM-mediated but also adhesion mediated by other molecules such as cadherin and L1 (Fujimoto et al., 2001; Rutishauser and Landmesser, 1996). In addition, PCA-NCAM is causally related to synaptic plasticity and structural remodeling (Dityatev et al., 2004; Muller et al., 1996). The PSA-NCAM increase thus suggests an attenuation of synaptic adhesion in the motor cortex, leading to a higher remodeling rate. The increase was evident in some animals even just 3 days after SCI, possibly suggesting that the PSA-NCAM increase is not only associated with formation of new synaptic structures but also removal of obsolete ones as part of the elimination process. We observed dynamic changes in the density and morphology of spines during a 4 week-period. Immunoblot analysis of PSA-NCAM expression provided a more detailed temporal profile; the increase was observed until 2 weeks after SCI, and its expression returned to control value by 4 weeks, suggesting that synaptic remodeling after SCI proceeds with a time scale ranging from days to weeks, but not in the order of months. Thus, SCI may initiate a period with a higher capacity for structural remodeling of synapses in the motor cortex for an initial few weeks after injury. This notion is consistent with a recent report that the efficacy of rehabilitation after stroke declines when instituted more than 14 days after injury (Biernaskie et al., 2004). The process of synaptic remodeling may be followed by axonal remodeling in the intracortical connections or corticospinal projections that occurs with a more protracted time course like in the order of months to years (Florence et al., 1998; Weidner et al., 2001). It is possible that distinct neural mechanisms underlying plasticity take part in functional recovery with different temporal windows (Little et al., 1999). Therefore, defining a temporal window for each neural mechanism would help to develop proper rehabilitation strategies along the course of recovery. In summary, this study provides evidence that dynamic changes in density and morphology of dendritic spines underlie reorganization of synaptic connectivity in the motor cortex following SCI. This remodeling process seems to be accompanied by attenuation of synaptic adhesion with a time scale ranging from days to weeks, during which synapse elimination and formation of new synaptic connections may occur with a

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distinctive time course. The increase in spine length may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI. Modulation of this process such as increasing the extent of remodeling or extending its temporal window may be a future therapeutic strategy to enhance functional recovery following SCI. Acknowledgments We thank Dr. Bogdan Stoica for technical advice for confocal microscopy, Dr. Zhanyan Fu for assistance in DiI labeling, and Dr. Barry Wolfe and Dr. Robert Yasuda for generous supply of NMDA receptor subunit antibodies and technical comments on the immunoblot experiment. We also thank Dr. Thomas Finn and Dr. Daniel Pak for helpful comments on the manuscript, and Dr. Shibao Feng for statistical consultation. This study was supported by NIH grant NS27054. References Adkins, D.L., Bury, S.D., Jones, T.A., 2002. Laminar-dependent dendritic spine alterations in the motor cortex of adult rats following callosal transection and forced forelimb use. Neurobiol. Learn. Mem. 78, 35–52. Annis, C.M., O'Dowd, D.K., Robertson, R.T., 1994. Activity-dependent regulation of dendritic spine density on cortical pyramidal neurons in organotypic slice cultures. J. Neurobiol. 25, 1483–1493. Bareyre, F.M., Kerschensteiner, M., Raineteau, O., Mettenleiter, T.C., Weinmann, O., Schwab, M.E., 2004. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat. Neurosci. 7, 269–277. Benson, D.L., Schnapp, L.M., Shapiro, L., Huntley, G.W., 2000. Making memories stick: cell-adhesion molecules in synaptic plasticity. Trends Cell Biol. 10, 473–482. Biernaskie, J., Chernenko, G., Corbett, D., 2004. Efficacy of rehabilitative experience declines with time after focal ischemic brain injury. J. Neurosci. 24, 1245–1254. Bregman, B.S., Goldberger, M.E., 1983. Infant lesion effect: II. Sparing and recovery of function after spinal cord damage in newborn and adult cats. Brain Res. 285, 119–135. Bregman, B.S., McAtee, M., Dai, H.N., Kuhn, P.L., 1997. Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat. Exp. Neurol. 148, 475–494. Bregman, B.S., Coumans, J.V., Dai, H.N., Kuhn, P.L., Lynskey, J., McAtee, M., Sandhu, F., 2002. Transplants and neurotrophic factors increase regeneration and recovery of function after spinal cord injury. Prog. Brain Res. 137, 257–273. Bruehlmeier, M., Dietz, V., Leenders, K.L., Roelcke, U., Missimer, J., Curt, A., 1998. How does the human brain deal with a spinal cord injury? Eur. J. Neurosci. 10, 3918–3922. Burns, S.P., Golding, D.G., Rolle Jr., W.A., Graziani, V., Ditunno Jr., J.F., 1997. Recovery of ambulation in motor-incomplete tetraplegia. Arch. Phys. Med. Rehabil. 78, 1169–1172. Cheng, H.W., Rafols, J.A., Goshgarian, H.G., Anavi, Y., Tong, J., McNeill, T.H., 1997. Differential spine loss and regrowth of striatal neurons following multiple forms of deafferentation: a Golgi study. Exp. Neurol. 147, 287–298. Clark, S.A., Allard, T., Jenkins, W.M., Merzenich, M.M., 1988. Receptive fields in the body-surface map in adult cortex defined by temporally correlated inputs. Nature 332, 444–445. Comery, T.A., Harris, J.B., Willems, P.J., Oostra, B.A., Irwin, S.A., Weiler, I.J., Greenough, W.T., 1997. Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits. Proc. Natl. Acad. Sci. U. S. A. 94, 5401–5404.

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