A total of 41 adult Sprague-Dawley were used in this study. The different experimental groups are indicated in . In 32 animals we used western blot analysis to assess whether passive hindlimb bike training after thoracic transection of the spinal cord affected the expression in the somatosensory cortex of proteins associated with plasticity (). In 9 additional animals we performed single-neuron mapping in the deafferented somatosensory cortex at the end of the study to electrophysiologically verify the presence of exercise-dependent cortical reorganization (). All procedures were performed under the guidelines of the National Institutes of Health, and approved by the Institutional Animal Care and Use Committee of Drexel University.
Spinal Cord Transection
Animals received a complete thoracic transection of the spinal cord with procedures that are similar to our previous studies 
. Briefly, adult female Sprague-Dawley (Charles River) rats were anesthetized with isoflurane (2–3% with oxygen) and the spinal cord was exposed by laminectomy at the T8/T9 level. The cord was transected with iridectomy scissors followed by aspiration of tissue within the cavity. A collagen matrix, Vitrogen, was injected into the site of the transection to fill the cavity. The muscle and skin were sutured in layers with 5-0 silk. Animals were then warmed, and when they became active, returned to their home cages. Bladders were manually expressed until the animals were able to void on their own. Animals were housed under a 12 h light/dark cycle (lights on at 07
00) with free access to food and water.
Passive Hindlimb Bike Exercise
As in previous studies, hindlimb bike exercise consisted of two 30-minute sessions with a 10-minute break, 3 days per week (M, W and F) 
. This exercise regimen involved suspending the rats on a sling with the hindlimbs hanging down and the hind feet strapped onto the pedals of a bicycle-type device that was driven by a motorized belt. The exercise consisted of a pedaling motion that flexed one limb while extending the other without overstretching the limbs. Cycling speed was 0.5 Hz. This was, therefore, a passive exercise of the hindlimbs only. Animals did not watch the exercised hindlimbs. Sham exercise consisted of placing the animals on the bike for 70 minutes, 3 days per week, but without moving the pedals.
Western Blot Analysis
Immediately following their last exercise session, animals were anesthetized with isoflurane and then decapitated. The brain and spinal cord were immediately removed, sliced on ice into 2 mm thick coronal slices and flash-frozen on dry ice. We focused on the sensorimotor cortex. Each tissue sample was placed into a FastPrep 120 tube (MP Biomedicals, Irvine, CA) in cold homogenization buffer and homogenized with FastPrep 120 for 40 sec, centrifuged at 1000 g for 10 min, transferred into 1.5 ml microfuge tubes and centrifuged at 20,000 g for 15 min. Protein concentration was assessed by using BioRad DC protein assay method (BioRad, Hercules, CA). Homogenates were fast frozen on dry ice and stored at −80°C until further processing. Proteins were run on pre-cast 26 well SDS gels (BioRad) either 10% or 4–15% gradient, depending upon expected protein molecular weight. Protein standards (Biorad) were used to determine protein size. For each antibody an initial amount of 30 µg total protein/well was loaded at four different dilutions (1
10). Once the optimal dilution was identified, it was held constant across all samples. Gelled proteins were transferred to 0.2 µ nitrocellulose membrane using I Blot system (Invitrogen). After transfer the membrane was incubated for 1 hr at room temperature in Li Cor western block (Li Cor, Lincoln NE) solution and then overnight at 4°C in primary antibody solution (Li Cor Block +0.1% Tween) with one of the following primary antibodies: adenylate cyclase isoform 1 (ADCY1; Bioworlde BS2391), brain derived neurotrophic factor (BDNF; Santa Cruz SC-20981), tyrosine kinase B (trkB; Millipore 07–225), 43 kD growth associated protein (GAP43; Epitomics 2259-1), leucine-rich repeat and Ig domain-containing Nogo receptor-interacting protein (LINGO-1; Santa Cruz SC-134597), Cyclin-dependent kinase 5 activator 1 (p35; Bioworlde BS2065). Antibody anti Beta-actin (Sigma A5441) was added to the incubation solution for endogenous control. After incubation in primary antibodies the blots were rinsed 3×10 min in PBS +0.1% Tween and incubated 1 hr and RT in block solution containing the following secondary antibodies developed in goat: anti-rabbit IgG IRDye700DX-conjugated, cat #611-130-122 and anti-mouse IgG IRDye800DX-conjugated, cat #610-132-121 (Rockland, Gilbertsville, PA) and HRP conjugated streptactin (BioRad 161-0382), then washed 3×10 min in PBS+Tween and rinsed in PBS. Processed membranes were exposed on Li-Cor Odyssey infrared imager at both 700 and 800 nm wavelengths for appropriate exposure times. All protein level values were obtained on the gel image by subtracting the background intensity from the signal for the protein of interest. The same procedure was performed for the control protein (Beta-actin) and each blot was normalized to its corresponding actin value. For statistical and illustration purposes, all western blot data for each animal and each protein were divided by the average value of the corresponding protein in the normal animals group and expressed as percentage. Note that in this way the average value for normal animals is 100% but the variability between animals (see error bars) is maintained.
Acute single-neuron mapping of the deafferented hindpaw cortex was performed at the end of the study with similar techniques as in our previous study 
. Rats were anesthetized by intraperitoneal injection of urethane anesthesia (1.3 g/Kg) and placed in a stereotaxic frame. Craniotomies were performed over either the right or left cortex to expose the hindlimb representations in the primary somatosensory cortex. The stereotaxic coordinates for hindlimb craniotomy were from 0 to 3 mm posterior to bregma and from 2 to 3 mm lateral 
. Electrode penetrations were defined using the stereotaxic coordinates for the hindlimb somatosensory cortex 
. For all animals, the anesthesia level was maintained at Stage III-4 
A high impedance (10 MΩ) tungsten microelectrode (FHC, Inc, Bowdoin, ME) was mounted on a stereotaxic electrode manipulator. A ground wire was inserted into the brain adjacent to the craniotomies. The microelectrode was then moved to the anterior-posterior and medial-lateral coordinates that defined a predetermined location above the hindlimb somatosensory cortex, and lowered, perpendicular to the surface of the brain, to penetrate the dura and pia. The microelectrode was then slowly inserted into the brain.
The signals from the microelectrode were continuously monitored on the oscilloscope and audio speakers as the electrode was lowered. When a neuron was encountered, the dorsal/ventral coordinates of the cell were noted. Two experimenters then determined whether the identified cell responded to sensory stimulation. The first experimenter, with knowledge of the electrode placement, used wooden probes to touch the hair/skin on the forelimb and shoulder. The second experimenter, blind to the position of the electrode and treatment group of the animal, determined if the cell responded to the stimulus, predominately by listening for a change in firing rate. If the cell did not modulate its firing rate in response to the stimulation, the cell was noted as negative. If the cell did modulate its firing rate, the cell was noted as positive. If the cell was noted as positive, then the receptive field of the cell was identified by tapping locations on the body rostral to the level of the injury. Stimulation of any body surface that modulated the cell’s firing rate was considered part of the cell’s receptive field. To ensure that tapping forces between animals and across sites were uniform, the responses elicited by the wooden probe were periodically compared to responses elicited by von Frey filaments to calibrate the stimulus applied by the wooden probe. The stimulation consisted of pressing a filament gently against the skin, perpendicular to its surface until the filament bent 90 degrees. This procedure was done 5 times for each filament and skin site, to ensure reproducibility of the results. The filament necessary to elicit a response similar to the wooden probe was noted and compared across animals and locations. The filaments required to produce an equivalent response ranged from 4.31 (bending force of 2 g) to 4.93 (bending force of 8 g) across animals and animal groups. There were no identifiable differences in the distribution of filaments used between the groups.
After a cell was characterized, the microelectrode was moved at least 50 microns deeper (with respect to the cortical surface) before another cell could be identified in the same penetration to ensure a new cell was encountered. For every cell identified, the stereotaxic coordinates of the microelectrode position were identified allowing us to evaluate neuronal responsiveness for each layer of the cortex. To minimize tissue damage and its possible effects on cell responsiveness during later penetrations, no more than 6 penetrations were performed per animal.
Perfusion and Histological Processing of the Brain and Spinal Cord
At the end of the mapping sessions, the rats were perfused transcardially with buffered saline, followed by buffered 2% paraformaldehyde, and then by buffered 2% paraformaldehyde containing 10% sucrose. The cortex was removed and flattened between two glass slides. The tissue was cryoprotected in 30% sucrose and sectioned (70 microns parallel to the pial surface) frozen on a sliding microtome. Series of these sections from the cortex were stained for cytochrome oxidase (CO) activity 
. Spinal cords were removed and placed in phosphate buffer containing 30% sucrose for 72 h. Specimens were frozen in OCT and sectioned on a freezing microtome at 20 µm. The transection segments of the spinal cords were sectioned parasagitally, and alternate sections were Nissl-myelin stained. The resulting sections were examined under a microscope to confirm completeness of the transection.
To assess the effects of spinal cord transection alone on plasticity-related proteins in the sensorimotor cortex, we compared ‘sham-exercise’ transected animals to normal animals by entering the data into a two-way independent-measures analysis of variance (ANOVA). The factors were ‘protein’ (ADCY1, BDNF, trkB, GAP43, LINGO-1 and p35), and ‘time from lesion’ (normal, 1 week after transection, 8 weeks after transection). In case of significant interaction, we performed follow-up one-way ANOVAs on individual proteins, followed by Fisher’s post-hoc test (as in Endo et al., 2007).
To assess the effects of passive hindlimb bike exercise after spinal transection on plasticity-related proteins in the sensorimotor cortex, the data from ‘bike-exercise’ and ‘sham-exercise’ animals were entered into a three-way independent-measures analysis of variance (ANOVA). The factors were ‘exercise’ (bike or sham), ‘protein’ (ADCY1, BDNF, trkB, GAP43, LINGO-1 and p35) and ‘weeks’ (1 or 8 weeks). In case of significant interactions, we performed follow-up two-way ANOVAs on individual proteins.
To assess the effects of passive hindlimb bike exercise after spinal transection on the electrophysiological reorganization of the deafferented cortex, we performed two types of analyses. First, the percentage of cells stereotaxically located in the deafferented hindlimb cortex that responded to stimulation of the intact forelimb were compared between bike animals and sham animals using two-proportion tests, separately for all cells and for each cortical layer. Second, the percentage of cells per track stereotaxically located in the deafferented hindlimb cortex that responded to stimulation of the intact forelimb were entered into a two-way mixed ANOVA, considering each track as an independent sample. The factors were exercise (bike or sham, independent measures) and cortical layer (supragranular, granular, infragranular, repeated measures).
Results were considered significant at p<0.05.