Animals were obtained from a supplier accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Kittens were delivered in litters along with a lactating mother at postnatal day 28. Columbia University and the New York State Psychiatric Institute Institutional Animal Care and Use committees approved all experimental procedures. Three groups of animals were compared: controls (CST terminations: n=3; behavior: n=2), unilateral inactivation (CST terminations: n=3; behavior: n=6 for impaired and unimpaired sides), and inactivation and stimulation (CST terminations: n=6; behavior: n=4).
We made every effort to make the general behavioral experiences of the three animal groups as similar as possible. All animals were raised in a highly enriched multilevel cage, with constant access to cat toys and facility and laboratory personnel. Inactivation only and inactivation/stimulation animals were raised during the entire experimental period, which included the period of stimulation and behavioral testing, with at least one other littermate and the mother. Moreover, our published findings on the effects of unilateral inactivation (Friel and Martin, 2005
; Friel et al., 2007
) examined animal performance on the ladder task daily, between weeks 7-11. Despite this intensive daily enrichment, these animals did not show anatomical or behavioral improvement. Overall, no differences were observed in anatomical or behavioral outcome as a result of subtle differences in day-to-day experiences.
Sensory-motor cortex activity blockade
To block neuronal activity in forelimb primary motor cortex (M1), we infused the GABAA
agonist muscimol (10 mM in sterile saline; Sigma Aldridge, St. Louis, MO) using a 28-ga hypodermic needle cannula (Durect Corp., Cupertino, CA)(Martin et al., 1999
). The cannula was connected with vinyl tubing (Scientific Commodities; Lake Havasu City, AZ, size 4) to an osmotic minipump (Alzet, model 2002; Durect Corp.) filled with the muscimol solution. Using the metabolic marker cytochrome oxidase, this infusion maximally inhibits a 2.5-3 mm radius of cortex at the infusion site and has a smaller effect for an additional 4-5 mm radius (Martin et al., 1999
)(i.e., total spread of inactivation, 6.5-8 mm). Inactivation reduces immunostaining of parvalbumen, a marker of activity that is also activity-dependent (Friel et al., 2007
), to a similar extent.
For cannula and pump implantation, animals were administered atropine (0.04 mg/kg i.m.) to reduce tracheal secretions. A mixture of Acepromazine (0.03 mg/kg i.m.) and Ketamine hydrochloride (32 mg/kg i.m.) was given to induce anesthesia. The trachea was intubated and the cats were maintained in an areflexive condition during surgery on 1%-2% isoflurane. Animals were given a broad-spectrum antibiotic (Cefazolin) at the time of surgery and an analgesic (Buprenorphine) afterwards.
A 5 mm wide small craniotomy was made over the forelimb area of M1 (Martin, 1996
; Chakrabarty and Martin, 2000
). The cannula was inserted 2 mm below the pial surface and was fixed to the skull with screws and dental acrylic cement. The pump, which was implanted in a subcutaneous pocket between the shoulder blades, delivered muscimol at the rate of 0.5 μL/hr during postnatal weeks 5-7. At the end of the infusion period, the cannula was cut from the pump to terminate infusion. We verified that the animals receiving stimulation after inactivation had the same behavioral impairments during and after cessation of the inactivation. All animals in this study showed abolished or substantially impaired contact placing during the infusion period from weeks 5-7, as in our previous studies (Martin et al., 1999
; Friel et al., 2007
Pyramidal tract electrode and electrode implantation
As in our previous study (Salimi and Martin, 2004
), we used an array of Teflon insulated stainless steel microwires (50 μm diameter; A-M Systems, Inc.; Carlsborg, WA) for stimulation. The microwires (four wires staggered in length by 250 μm) were attached to a stiff tungsten wire (500 μm outer diameter, rounded at the end) using Carbowax (Fisher Scientific, Pittsburgh, PA) (Palmer, 1978
). The Carbowax attachment method permitted detachment of the wires when they were within the brain because it is soluble in a warm aqueous environment; microwires were detached from the carrier above the pia by rinsing the assembly with warm sterile saline.
The microwire electrode array was implanted into the left pyramidal tract (PT) two days after terminating the infusion, and in one animal, one month. Positioning of the electrode array was guided by our previous studies in kittens of the same age (Meng et al., 2004
; Salimi and Martin, 2004
). The array was inserted stereotactically into the caudal medulla, between 16° and 20° posterior to anterior angulation. The array was positioned 0.5 mm lateral and -4 to -6.00 mm posterior to interaural zero. The array was driven to the skull floor and then raised 0.5 mm to allow for the distance between the skull and the pyramid. A silver wire attached to a cranial bone screw was used as the indifferent. The microwires, together with the connector, were fixed in place using cyanoacrylate and dental acrylic cement. We confirmed the position of the electrodes post mortem with the Prussian blue histochemical reaction to stain iron deposits at the stimulation site (see Supplemental Figure 1A
) and by locating marking lesions made just prior to euthanizing the animals on Nissl-stained sections. The day after electrode implantation, we determined the threshold for evoking a motor response from each microwire individually (see below for threshold testing method). We chose one of the microwires as the stimulating electrode based on the lowest threshold for evoking contralateral forelimb movements.
Corticospinal axons tracing
Four weeks before euthanizing each animal, we pressure injected (Picopump, WPI, Sarasota, FL) the anterograde tracers biotinylated dextran amine (BDA; Invitrogen, Inc., Eugene, OR; 10% in PBS) into the forelimb area of M1 of one hemisphere and Lucifer Yellow-dextran amine (LY-DA; Invitrogen, Inc., 1% in PBS) into the other. In each M1, we made three tracer injections, 1.5 mm below the pial surface (300 nL each, separated by 1-1.5 mm). For determining CST projections from inactivated M1, tracer injections were made 3-7 days after cessation of the infusion. The three injections were positioned rostral, lateral, and caudal to the muscimol infusion site, as in our previous studies (Friel and Martin, 2007
). This ensured that we were examining the previously silenced projection. BDA was used to label the initially inactivated cortex in all but one animal. Injections in the M1 contralateral to the inactivation were made in locations homotopic to the injections in the inactivated M1. Uptake and/or anterograde transport of BDA are not dependent on neural activity. To control for this concern in our previous study and to examine the effects of activity blockade after the critical period, we injected BDA into M1 at week 7 several days prior to the onset of muscimol infusion (Friel and Martin, 2005
). The animal was perfused at week 11, after one month of inactivation and tracer transport. The pattern of CST axon labeling in this animal was identical to the pattern of axonal labeling in age matched controls. Also in that study (Friel and Martin, 2005
), we found similar termination patterns irrespective of injecting tracers one week before infusion (trace from 4-7 weeks; inactivate between weeks 5-7) or immediately following (inactivate between weeks 5-7; trace from 7-10 weeks). The pattern of CST axon terminal labeling using LY-DA was always the same as with BDA. Finally, effective intracortical transport occurred during local cortical activity blockade by tetrototoxin (Rhoades et al., 1996
A constant current stimulator (model 2100; A-M Systems, Inc) delivered 45 ms trains of stimuli (330 Hz, 20 μsec duration biphasic pulses), 2-3 hours a day, once every 3 seconds, for 21 days at the end of the inactivation period. Average total stimulation time was 2.6 hours/day. Each day we used a current that was just sufficient to evoke a contralateral forelimb movement for each stimulus train. To determine the threshold, we started at zero and increased the current in 5-10 μA intervals until a response was observed. Next we raised the current an additional 20-30 μA and then reduced the current in small steps and noted the value when movements were no longer evoked. The threshold was the average of the currents just evoking a movement during the ascending and descending series. The animals were typically playful while determining the threshold, sometimes making determination difficult. To ensure that we did not underestimate threshold during this period (e.g., the animal's voluntary response was interpreted as a stimulus-evoked response), and risk stimulating below threshold, we sometimes raised the current 5-10 μA. Once the current value was established, the animal was placed in an enclosure for the stimulation period and checked every 10-15 minutes. Animals usually slept for the duration of the stimulation period.
Tissue Preparation and Staining
At the end of the survival period, animals were deeply anesthetized with sodium pentobarbital (30 mg/kg i.v.), injected with heparin (500 units i.v.) and perfused transcardially with warm saline (39°C). This was followed by 4% paraformaldehyde (in 0.1 M PB). The brain and spinal cord were removed, postfixed if necessary and transferred to 20% sucrose in 0.1 M PB overnight. Transverse frozen sections (40 μm) through the C7–T1 spinal cord were cut. Alternate sections were processed for BDA histochemistry and LY-DA immunocytochemistry. Alternate transverse sections were cut through the medulla for Nissl and Prussian blue staining to identify the location of the electrode array tip and marking lesion. Prussian blue permits visualization of iron deposits at the tip of the electrode, which is the result of the passage of current (see Supplemental Figure 1A
). Sagittal sections were cut through the cortex in both hemispheres to identify the tracer injection sites.
For BDA visualization, sections were incubated in PBS containing 1% avidin-biotin complex (ABC kit; Vector Labs, Burlingame, CA) according to the manufacturers specifications. Sections were incubated with the chromogen diaminobenzidine (DAB; Sigma, St. Louis, MO) for 6-30 min. For visualization of LY-DA, sections were incubated at 4° overnight in PBS containing 0.01% rabbit anti-LY antibody (Invitrogen Carlsbad, CA) in blocking buffer (3% goat serum in 1× PBS with 0.2% Tween). After rinsing again, sections were incubated for 2 h at room temperature in blocking buffer containing 0.2% anti-rabbit antibody conjugated to peroxidase. After a final rinse, sections were incubated with the chromogen diaminobenzidine (DAB) for 5-30 min.
Anatomical data acquisition and analysis
We traced labeled CS axons (terminal and preterminal axons) and marked the locations of axon varicosities, which are putative presynaptic sites. Axon varicosities were defined, as in our previous study, as a region of the axon that is at least three times axonal width (Li and Martin, 2002
). Care was taken to verify that axon varicosities were not kinks or other irregularities in the axon. Using laser scanning confocal microscopy, we have found that CS axon varicosities colocalize the synaptic vesicle protein synaptophysin (Meng et al, 2004
) and appose sites containing puncta of the postsynaptic marker PSD-95 (D. Trachtenberg and J. Martin, unpublished observations). Transmission electron microscopy confirmed the confocal observations that the CS axon varicosities are filled with synaptic vesicles and appose sites on the postsynaptic membrane with a postsynaptic density (G. Holstein and J. Martin, unpublished observations). Thus, CS axon varicosities, as defined in this study, are likely to mark presynaptic sites, either en passant or terminal. We traced labeled CS axon terminations and varicosities in gray matter of spinal cord using the program Neurolucida (MBF Bioscience, Williston, VT). LY-DA and BDA labeled axons were traced from transverse spinal sections at 200× magnification. We also traced the gray matter boundary, Rexed's laminae (see below), and anatomical landmarks. Digital tracings of individual sections were corrected for orientation and aligned with one another according to fiduciary marks (intersection between the gray matter above the central canal and the dorsal median septum). The corrected digital images were exported and measured using custom programs written in MATLAB (MathWorks, Natick, MA).
To determine local changes in the amount of labeling, digital files of gray matter labeling on individual sections were divided into 52μm X 52μm square regions of interest (ROI). For each ROI, we computed the mean density of traced axons and marked varicosities. A matrix of mean axon or varicosity density was generated in MATLAB that preserved the mediolateral and dorsoventral dimensions of the distribution of label in the gray matter. Using this matrix, we generated topographic maps of the regional distribution of axonal label and varicosities, where local density is represented according to a color scale, from the lowest density (blue) to the highest (red). To compare the distribution of labeling across animals, we used a commercially-available program (Morpheus) for the Macintosh computer (Apple Computer) to “warp” axon distributions of each individual animal into a standardized spinal gray matter shape. We then averaged individual animal data from each group. Warping (sometimes termed morphing) systematically stretches the border of the starting image (i.e., from a particular animal) to match that of the final (i.e., average). In doing so, the internal spatial structure of the data are also stretched. This is a standard technique for minimizing spatial variability in human brain imaging, and we have used this technique previously for a similar purpose (Campos et al., 2008
). For this study, we use warping as a heuristic, rather than analytic, tool to allow the reader to better appreciate differences in axonal distribution between the animal groups in relation to the laminar organization of the spinal cord. Supplemental Figure S2
shows the original gray matter outlines for the warping; the final (i.e., warped) outline is shown in and . To quantify changes in the distribution of label across animals we determined the amount of label within Rexed's laminae. The following laminae were identified, using Rexed's original criteria (Rexed, 1954
), on Nissl- or counter-stained spinal cord sections. Laminae 1-3: lamina 3 contains uniformly small cells. Lamina 4: mixture of cell sizes, including very large cells not present dorsally; ventral border corresponded to a horizontal line from the top of the neck of the dorsal horn. Lamina 5: reticulated zone, with reduced cell density. Lamina 6: anatomically corresponds to the base of the dorsal horn (lamina 6 is more commonly included within the intermediate zone; see Results). Lamina 7: includes all ventral territories, exclusive of the medial and lateral motor pools. We distinguished a dorsal region, which corresponded to the portion dorsal to the motor pools, from a ventral region that separated the medial and lateral motor pools. Laminae 8 and 9: medial and lateral motor pools; identified by the presence of motoneurons. Local axon and varicosity density, as a proportion of labeling within each of the laminae, was determined using scripts written in Matlab and Microsoft Excel.
Figure 1 Stimulation after previous M1 inactivation redistributes CS terminations to intermediate and ventral motor laminae. Color coded topographic maps show the local density of axons (A) and varicosities (B) in representative animals. Warped and composite (average; (more ...)
Figure 3 Stimulation following inactivation reduces the density of ipsilateral corticospinal boutons but not axons. Similar format to ; representative animals are shown in A, B and, a composite average in C (inactivation, n=3; inactivation-stimulation, (more ...)
Behavioral training and testing
All animals were exposed to the laboratory environment several times a week, beginning between weeks 6/7. This enabled obtaining snapshots of performance during the stimulation period and ensured that animals were acclimatized to the environment when testing was done after cessation of stimulation. We examined animals performing a horizontal ladder task (Friel et al., 2007
). The ladder was made of Plexiglas (88 cm long, 18.4 cm wide; 9 mm square wide rungs placed every 5.8 cm), with stationary platforms at either end. Cats were placed at one end of the ladder, and meat cubes were placed at the other end. During testing, the cat walked across the ladder from the start platform to the food reward. To prevent cats from memorizing rung position, we placed them at different positions on the platform for each trial while keeping the distance between rungs constant. This resulted in their starting to step on the rungs with either forelimb. Moreover, the first ladder rung to be stepped on differed from trial-to-trial. Animals were first introduced to the task during daily sessions of approximately 10-15 minutes and, once acclimated to the lab and the testing apparatus, most animals were tested twice weekly.
Analysis of behavioral performance
Videotapes of testing sessions were imported into a video editing program (iMovie; for the Apple Macintosh computer) and analyzed for forelimb kinematics to measure endpoint control. We used the same analysis criteria as in our previous studies (Friel et al., 2007
). The forelimb contralateral to inactivation/stimulation is termed the affected limb and the other, the unaffected limb. We showed that performance of the unaffected limb was identical to that of animals that did not receive muscimol infusion (i.e., untreated control) or received only infusion of the saline vehicle)(Friel et al., 2007
). We measured the distance that the tip of the cat's forepaw extended beyond the edge of the rung of the ladder (termed forward distance). We used the program ImageJ, in combination with the program Afloat (Window management tricks for Mac OS X, Microsoft Visual Studio, Inc) on the Apple Macintosh computer to measure forward distance. Distance measures from the computer screen were converted to cm by scaling according to a calibrated distance on each video file. Trials were not scored if the movement was halted during the step, the movement changed direction, or the animal jumped. Images from the video files were analyzed at 30 Hz. Performance of the inactivation/stimulation animals was compared with data from animals receiving inactivation only, which were reanalyzed from previous studies (Friel and Martin, 2007
; Friel et al., 2007
We used the program Statview for the Apple Macintosh computer to determine the statistical significance of differences. Standard and repeated measures ANOVA were used to compare the dorsoventral distributions of labeling in the inactivation only and inactivation/stimulation groups. Post hoc testing was used to determine if particular levels of the spinal cord, known to be relevant to control by the corticospinal terminations, showed significant differences. Pair-wise t-tests were also used, with correction for multiple comparisons. Linear regression was used to assess significance of changes in threshold currents with stimulation. Unpaired t-tests were used to compare forelimb movement endpoint errors for the inactivated and inactivated/stimulation groups.