Animals

Adult (7- to 8-week-old) female and male C57BL/6J mice (stock no. 000664, The Jackson Laboratory) were used for all experiments, except those specifying different developmental stages and GFP-M mice. GFP-M (stock no. 007788; RRID:IMSR_JAX 007788) mice expressing GFP under the control of Thy1 promoter were purchased from The Jackson Laboratory. Mice were randomly assigned to experimental groups. Experimenters were blind to group assignment and experimental conditions.

Antibodies

The following antibodies were used: mouse monoclonal anti-βIII tubulin (Tuj1) (801202, RRID:AB_10063408, BioLegend), rabbit polyclonal anti-βIII tubulin (T2200, RRID:AB_262133, Sigma-Aldrich), mouse monoclonal anti-NeuN (MAB377, RRID:AB_2298772, Millipore), Alexa Fluor 488 Phalloidin (A12379, RRID:AB_2315147, Invitrogen), rabbit polyclonal anti-α2δ2 (ACC-102, RRID:AB_11124467, Alomone Labs), rabbit monoclonal anti–c-Fos (9F6) (2250S, RRID:AB_2247211, Cell Signaling Technology), rabbit polyclonal anti-GFAP (Z0334, RRID:AB_10013382, Dako), rabbit polyclonal anti-mCherry (ab167453, RRID: AB_2571870, Abcam), rabbit monoclonal anti-PKC gamma (59090, RRID: AB_2799557, Cell Signaling Technology), chicken polyclonal anti-Homer1 (160006, RRID:AB_2631222, Synaptic Systems), guinea pig polyclonal anti-VGLUT1 (135304, RRID:AB_887878, Synaptic Systems), rabbit polyclonal anti–Caspase-3 (ab 13847, RRID AB_443014, Abcam),and chicken polyclonal anti-GFP (GFP-1020, RRID:AB_10000240, Aves Labs).

Primary neuronal cultures

Cortical neuronal cultures were derived from cortices of embryonic 17.5-day-old mouse (C57BL/6J) embryos. Cortices were extracted, dissociated, and cultured as previously reported (63). Briefly, cortices were minced and dissociated in the same buffer with 1,800 U/ml trypsin at 36.5°C for 20 minutes. Next, 200 U/ml DNase I and 3,600U/ml soybean trypsin inhibitor were added to the suspension, and cells were triturated through a 5-ml pipette. The tissue was allowed to settle for 5–10 minutes, and then the supernatant was collected, and the remaining tissue pellet was retriturated. The combined supernatants were centrifuged through a 4% BSA (A3059, Sigma-Aldrich) layer and the cell pellet was resuspended in neuronal seeding medium (NSM), which consisted of Neurobasal Medium (12348017, Life Technologies) supplemented with B27 (17504044, Life Technologies). The culture was maintained in a humidified atmosphere containing 5% CO 2 in air at 36.5°C. To achieve Cacna2d2 overexpression, dissociated cortical neurons were electroporated (program: CA138, NC0301987, Fisher Scientific) with a mixture of GFP (2.5 μg, pmaxGFP, Lonza) plus either Cacna2d2 (4 μg, MC223740, Origene), Cacna2d2(R282A) (17) or empty (4 μg) plasmid DNA. Electroporated neurons were then plated at low density on coverslips coated with poly-D-lysine (P6407, Sigma) and the electroporation medium was replaced with fresh medium (with or without GBP) 2 hours after plating.

Corticospinal neurons retrograde labeling and immunohistochemistry

A C3–5 laminectomy was performed in GFP-M adult mice, and Fluoro-Gold tracer (1%, Fluorochrome) was injected (0.5 μl/spot at 0.1 μl/min, 4 spots) into the dorsal corticospinal at the C3–5 spinal cord. After 3 days, the mice were perfused and the brains dissected and sequentially dehydrated in 10%, 20%, and 30% sucrose. Tissues were then embedded in optimum cutting temperature (OCT) compound (Tissue-Tek), frozen, sectioned (20- to 30-μm thick, HM525 NX, Thermo Fisher Scientific) and mounted on slides. Slides were warmed at 37°C for 30 minutes and OCT was washed away with PBS. Sections were then blocked at room temperature with 2.5% bovine serum albumin (A3059, Sigma-Aldrich) in PBS with 0.1% Triton-X100 for 1 hour and incubated overnight at 4°C with the primary antibody. After washing 3 times with PBS, sections were incubated with Alexa Fluor–conjugated secondary antibodies (1:500, Life Technologies). When necessary, sections were counterstained with DAPI (1:10,000, D9542, Sigma-Aldrich). Images were taken using a confocal (SP8, Leica and C2 plus, Nikon) or epifluorescence microscope (Axio Observer Z1, Zeiss) and linear fluorescence intensity was calculated using Photoshop (version 20.0.1, Adobe) or ImageJ after background subtraction. The expression of α2δ2 (1:400) in Fluoro-Gold–labeled corticospinal neurons was measured by using Zen Blue software (Zeiss). A minimum of 3 independent biological replicates (2 or more sections/mouse) was analyzed per condition.

Immunoblot analysis

For immunoblotting, cultured embryonic cortical neurons and dissected sensory-motor cortices from mice at different stages of postnatal development were each lysed on ice in RIPA buffer (500 mM Tris-HCl pH 4.8, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing phosphatase (04906845001, Sigma-Aldrich) and protease inhibitors (5892791001, Sigma-Aldrich), centrifuged, and the supernatant collected. Using Bradford reagent (Bio-Rad), the protein concentration of the lysate was determined, and a portion of the lysate (10–20 μg total protein) was then fractionated by SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a nitrocellulose membrane (0.2 μm, Bio-Rad), which was stained to confirm equal loading and transfer of the samples with Ponceau S (P7170, MilliporeSigma). After blocking at room temperature with 5% nonfat milk (170-6,404, Bio-Rad) in TBST for 1 hour, the membrane was then probed with a rabbit polyclonal anti-α2δ2 (1:1,000). Rabbit polyclonal anti-Tuj1 (1:20,000, Sigma-Aldrich) antibody was used as protein-loading controls. Densitometry analysis was done using ImageJ (NIH). The regions of interest that contained the bands to quantify had the same size across the immunoblot. After background subtraction, the intensity of α2δ2 bands at 130 and 105 kDa was measured and normalized to the loading control (e.g., Tuj1). To calculate α2δ2 expression, the 2 values were then summed. Three biological replicates for each experimental condition were analyzed.

RNA isolation

Total RNA was extracted from mouse cortices using the RNeasy kit (Qiagen) and cDNA was synthesized from 0.1–0.5 μg of RNA using random hexamers from the SuperScript VILO cDNA synthesis kit (11754050, Thermo Fisher Scientific). The subsequent cDNA was used in a real-time PCR (StepOne Plus, Applied Biosystem) using Fast SYBR Green Master Mix (4385612, Applied Biosystem). Melting curve reactions were run with each primer set. The β actin gene was used for normalization. The sequences of the primers used were as follows: Cacna2d2_s 5′-ACAAGGACAATCGGAACCTG-3′, Cacna2d2_as 5′-TGGGCTTTCTGGAAATTCTCT-3′, β actin_s 5′-ACAGCTTCACCACCACAGCTGA-3′, β actin_as 5′-GAGGTCTTTACGGATGTCAACGTC-3′. Normalized expression was calculated as dCt (gene norm) = Ct Cacna2d2 – Ct β actin and normalized expression = 2-dCt (gene norm)

In vivo recording of spontaneous firing

P14 and P28 mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight), and P7 with a mixture of ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight). A craniotomy was performed to expose the right sensory-motor cortex. A 32-channel silicon electrode array (Buzsaki 32-A32, NeuroNexus Technologies) connected to a stereotaxic frame was inserted 500 to 600 μm deep into the forelimb sensory-motor cortex. Spontaneous neuronal firing was recorded at a 25-kHz sampling rate and low-pass filtered at 10 kHz using the SmartBox acquisition system (NeuroNexus Technologies). Recording data were analyzed using Igor Pro (version 8, WaveMetrics). Briefly, data were filtered off-line at 300 to 3,000 Hz and smoothed using a sliding average of 5 points. Spikes were identified when passing the detection threshold of 4 times the baseline noise standard deviation (64–66). Detected spikes were then sorted into single units using the k-means clustering method after the principal component analysis (64, 66). The customized algorithm was scripted in Igor Pro and the experimenter manually reviewed sorted results.

Cervical spinal cord injury and corticospinal transduction

Mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). A C5 laminectomy was performed, and the spinal cord was crushed with no. 5 modified forceps (11254-20, Dumont, FST). The forceps were positioned to completely sever half of the spinal cord (Supplemental Figure 1D). Beginning 1 hour after injury, GBP (46 mg/kg body weight, PHR1049, CAS:60142-96-3, MilliporeSigma) or the corresponding volume of vehicle (0.9% sodium chloride, 0409-7138-09, Aqualite system) was administered (intraperitoneal injections, 3 times/day for the first week, 2 times/day until the end of the study). Three to four weeks before the end of the study, AAV particles for optogenetic or chemogenetic manipulation of neurons were injected into the right sensory-motor cortex (AP coordinates from bregma in mm: AP 1.0/1.3, 0.5/1.3, 0/1.3, –0.5/1.3, all at a depth of 0.6 mm from the surface, 500 nl/injection site). For immediate-early gene mapping, a cohort of mice with SCI was anesthetized, and electrical stimulation (300 μA, 0.5-ms biphasic pulse at 5 Hz for 15 minutes) of the right sensory-motor cortex was delivered using a tungsten concentric bipolar electrode (TM33CCINS, World Precision Instruments) connected to an isolated pulse stimulator (A-M systems Model 2100). The electrode was inserted at a depth of 0.5 mm from the surface (AP coordinates from bregma in mm: AP 0.25/1.3). At the end of each study (1 hour after stimulation for early gene mapping), mice were perfused with 4% paraformaldehyde. Sagittal (60-μm thick) and transverse (20- to 30-μm thick; 2.5 mm rostral and 6–7 mm caudal to the lesion) serial cryosections of the spinal cord were cut and collected to confirm the completeness of the lesion and tracing efficiency among experimental groups. Mice with incomplete lesions were excluded from the analysis. In mice injected with AAV-ChR2-eYFP, the portions of the unsectioned spinal cords containing the lesion site were cleared using the advanced CUBIC protocol (67) and imaged using a confocal microscope (SP8, Leica). The imaging software Imaris (version 9.2, Bitplane) was used for 3-dimensional rendering (Figure 4C). Three-dimensional images were used for quantification of regeneration using the ImageJ Simple Neurite Tracer plugin (68). Briefly, the contralateral side of the injured spinal cord was divided in 4 adjacent boxes (–1750/–1250 μm, –1250/–750 μm, –750/–250 μm, and –250/+250 μm from the lesion epicenter), each measuring 1,000 μm in width and 500 μm in length, and the ipsilateral side only contained 1 measuring box with the same size immediately below the lesion epicenter (0/+500 μm). Regenerating axons were then semi-automatically traced in each 3-dimensional box. For the contralateral side, the total length was calculated and divided by the number of corticospinal fibers counted at the level of medulla oblongata (c.a., 1 mm proximal to the pyramidal decussation) to account for variation in tracing efficiency in each mouse. The procedure to count corticospinal fibers at the medullary level was the same as described in Liu et al. (11). The axon regeneration values at different distances were displayed as a percentage of the average value of the vehicle group in all 4 boxes (Figure 4D). For the ipsilateral side, we calculated the number of traced corticospinal axons below the lesion site and normalized it to the number of labeled corticospinal axons at the medullary level. Immunohistochemistry was performed following standard protocols (see above, α2δ2 1:400, NeuN 1: 500, GFAP 1: 500, mCherry 1:300, GFP 1:500, PKCγ 1:200). Immunohistochemistry for c-Fos (1:1,000, Cell Signaling Technologies) required an antigen-retrieval procedure consisting of 2–3 minutes incubation of tissue slices in citric acid–based antigen-unmasking solution (H-3300, Vector Laboratories) at 95°C–100°C. The average density of c-Fos/NeuN positive cells within the spinal cord was then mapped using a 2-dimensional histogram. Standardized randomization and blinding strategies were adopted.

PTX

Adult mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight); at P10, they were anesthetized with a mixture of ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight). An incision was made at the left side of the trachea and a blunt dissection exposed the base of the skull where, to access the medullary pyramids, a craniotomy in the occipital bone was performed. The left pyramid was cut with a micro scalpel (no. 715, Feather) medially to the basilar artery and the wound was closed in layers with 6.0 (adult) and 9.0 (P10) sutures. The sham operation for the P10 cohort (sham and PTX surgeries were performed at P10) included a craniotomy in the occipital bone without cutting the left pyramid. The mice were placed on soft bedding in their home cage on a warming surface held at 37°C until awake and alert. After 2 weeks (PTX in adulthood) or 18 days (PTX at P10), the intact corticospinal tract was traced with 10% BDA (10,000 MW, D1956, Life Technologies). The mice were perfused with 4% paraformaldehyde 2 weeks after BDA injection. Spinal cords and medulla oblongata were post-fixed in 4% paraformaldehyde overnight at 4°C and then immersed in 10%, 20%, and 30% sucrose, and cryosections were prepared. After quenching endogenous peroxidase with 0.3% H 2 O 2 in PBS for 30 minutes, coronal sections were incubated for 2 hours with streptavidin–horseradish peroxidase conjugate (1:200 in 2% Triton PBS, NEL7500001EA, Perkin Elmer). The TSA Cyanine 3 system (SAT704A001EA, Perkin Elmer) was then used for immunofluorescence amplification of the BDA signal. The procedure to count corticospinal fibers at the medullary level was the same as described above. For quantification of sprouting corticospinal axons, the area of sprouting axons from the contralateral side was measured and divided by the total area of gray matter from the contralateral side (the detection threshold was set as 2 times the gray matter background signal of each individual image using ImageJ). This value was then normalized to the average value from the sham control (PTX at P10) or vehicle (PTX in adulthood) groups and presented as a sprouting index. Immunostaining was performed following standard procedure (PKCγ 1:200, NeuN 1:500, VGLUT1 1:1000, Homer1 1: 400, Caspase-3 1:300). Multidimensional surface reconstruction of putative synapses along BDA-labeled corticospinal axons was created using an ImageJ 3D viewer plugin (69). The imaging stack was filtered through a 2D Gaussian filter (radius: 2 pixels) to reduce signal noise. Surface reconstruction was then created for each channel. The image was then rotated to confirm presynaptic and postsynaptic alignment.

Immunocytochemistry and morphometric analysis

Mouse cortical neurons were grown on PDL-coated coverslips and fixed with 4% paraformaldehyde in 4% sucrose. Coverslips were then blocked at room temperature for 1 hour with 2.5% BSA and 0.1% Triton-X100 in PBS and incubated at 4°C overnight with the appropriate primary antibodies (Tuj1 1:1,000, Phalloidin 1:200, and α2δ2 1:400). After 3 rinses in PBS, the coverslips were incubated with Alexa Fluor–conjugated secondary antibodies (1:500, Life Technologies) and washed in PBS before mounting onto microscope slides. Fluorescence images were randomly taken with an inverted microscope (Axio Observer Z1, Zeiss) and analyzed using Zen Blue (Zeiss) and Fiji software (version 2.0.0-rc-54/1.5h) (70). This process was repeated for at least 3 independent experiments. The number of neurons quantified for each condition is indicated in the corresponding figure legend.

Optogenetics and in vivo multichannel recording of LFPs

AAV2/1-CamKIIa-hChR2(H134R)-eYFP (1-2e13 GC/ml, Addgene26969, Addgene) particles were injected into the right sensory-motor cortex. Four weeks after AAV injection, the mice were anesthetized and a laminectomy was performed to expose the cervical spinal cord between C3 and C6. The vertebral columns were stabilized by clamps attached to either side of the laminectomy site. A 32-channel silicon electrode (A4x8-5mm-50-200-177, NeuroNexus Technologies) connected to a stereotaxic frame was inserted at a depth of 500 and 700 μm into the spinal cord at the following locations: 500 μm rostral/ipsilateral, 500 μm caudal/ipsilateral, and 500 μm rostral/contralateral to the lesion site. A 473-nm diode-pumped solid-state laser (Shanghai Laser and Optics Century) was coupled with a 200-μm optical fiber, and the tip of the optical fiber was placed at c.a. 2 mm above the spinal cord rostral to the lesion site. Using a pulse waveform generator (Keysight, 33521B), the laser was set to deliver an approximately 25-mW 10-ms pulse every 5 seconds. The laser power was measured using a slide power sensor (S170C, Thorlabs) coupled with a laser power meter (PM100A, Thorlabs). The LFP was recorded at a 25-kHz sampling rate and low-pass filtered at 10 kHz using the SmartBox acquisition system (NeuroNexus Technologies). Recording data were analyzed using Igor Pro software (version 8, WaveMetrics). LFPs were low-pass filtered at 50 Hz. For each channel, 3–5 light-induced LFPs were averaged for quantification.

Chemogenetics

For chemogenetics experiments, corticospinal axons that project to the cervical spinal cord were transduced by injecting AAV2/1-hSyn-hM4D(gi)-mCherry (1-2e13 GC/ml, Addgene50475, Addgene) into the right forelimb sensory-motor cortex. Four weeks after AAV injection, the mice underwent horizontal ladder and cylinder behavioral tests starting 15 minutes after injection of CNO (1 mg/kg, i.p.) (4936, CAS: 34233-69-7, Tocris Bioscience) to transiently silence corticospinal projections. On the following day, the mice were administered an equal amount of vehicle (0.9% saline), and the behavioral procedure was repeated. The alternation of CNO and vehicle administration was repeated for 2 consecutive sessions for 1 week, and the results were averaged between sessions (Figure 5, G and H). Behavioral analysis was carried out by investigators blinded to the treatment.

Behavioral testing

Horizontal ladder. Before injury, mice were trained to walk across the ladder to an enriched cage in 1 direction. Baseline values were collected for all mice prior to injury. After the injury, and at regular intervals until the study endpoint, mice were placed at one end of the ladder and video recorded while moving across to the other end where the enriched cage was located. The recordings were then analyzed using VLC player, and the percentage of correct steps was calculated.

Toe spread index. Contralateral and ipsilateral forepaws were photographed (Sony Cybershot DSC-W800) at 117 dpi from the ventral surface. All images were taken when the mouse was bearing weight on a glass surface. Intermediary toe spread was established from the images by measuring the distance between the first and fourth toe using ImageJ software.

Cylinder test. The mice were placed in a 500-ml clear beaker with a small amount of bedding on the bottom. The mice were allowed to move independently and explore normally. The proportion of right versus left versus both paw placements on the side of the beaker was noted for 10 attempts and used to calculate the percentage of forelimb asymmetry. Baseline values were collected for all mice prior to injury and continued at regular intervals until the study endpoint.

Von Frey test. The mice were placed in a testing chamber, and the plantar threshold was measured using retractable monofilaments (Ugo Basile) and the “up-down” method. A quick withdrawal of the left hind paw was considered as a positive response. A pause of 20–30 seconds was given to allow sensory receptors to reach baseline levels between each monofilament application. The response threshold was calculated as the lowest force (in grams) that produced a retraction at least 50% of the time.

Activity box. The mice were placed in activity boxes (Columbus Instruments) for 10 minutes. Spontaneous activities in horizontal and vertical planes were recorded through compatible Fusion software (version 6.4 r1194, Omnitech Electronics). Baseline values were collected for all mice prior to injury and continued at regular intervals until the study endpoint.

For all behavioral tests described above, experimenters collecting and analyzing data were blinded to the treatment.

Statistics

Statistical analysis was performed using Prism (version 8.0.2; GraphPad Software) as follows: unpaired 2-tailed Student’s t test (Figure 1K; Figure 2, C, E, G, K, and M; Figure 3, C and E; Supplemental Figure 1F; Supplemental Figure 3B; and Supplemental Figure 5F), paired 2-tailed Student’s t test (Supplemental Figure 7, B and E), 1-way ANOVA followed by Dunnett post test (Figure 1F, Figure 3F, and Supplemental Figure 2B), 2-way ANOVA followed by Tukey’s post test (Figure 5E), 2-way ANOVA followed by Bonferroni’s post test (Figure 5, G and H). Trend tests and mixed models were performed using SAS (SAS 9.4; SAS Institute) as follows: linear trend test with log 2 transformation (Supplemental Figure 1A) or without log 2 transformation (Figure 1, D and H), mixed model with repeated measures using unstructured covariance matrix (Figure 4D), mixed model with repeated measures using compound symmetry covariance structure and controlled on baseline values (Figure 5, B and C, and Supplemental Figure 8, A–C), and mixed model with repeated measures (Supplemental Figure 8D). For all analyses performed, significance was defined as P < 0.05. Exact values of n and definition of measures are shown in the corresponding figure legends.

Study approval

All animal experiments were performed in accordance with and with the approval of the Institutional Animal Care and Use Committee at The Ohio State University.