DENDRITIC SPINE DYSGENESIS CONTRIBUTES TO HYPERREFLEXIA AFTER 1 SPINAL CORD INJURY 2

3 Abbreviated title: Dendritic Spine Dysgenesis in Hyperreflexia after SCI 4

5 Samira P. Bandaru, Shujun Liu, Stephen G. Waxman, and Andrew M. Tan 6

7 Department of Neurology and Center for Neuroscience and Regeneration Research, 8 Yale University School of Medicine, New Haven, CT 06510, and Rehabilitation 9 Research Center, Veterans Affairs Connecticut Healthcare System, West Haven, CT 10 06516 11 12 Corresponding author: 13 Andrew M. Tan, Ph.D. 14 Center for Neuroscience and Regeneration Research (127A) 15 950 Campbell Avenue, Building 34 16 West Haven, CT 06516 17 Tel: 203-932-5711 x3663 18 Fax: 203-937-3801 19 e-mail: andrew.tan@yale.edu 20 21 22 23 24 25 26 Conflict of Interest: None 27 28 29 30 31 32 33 34

Articles in PresS. J Neurophysiol (December 10, 2014). doi:10.1152/jn.00566.2014

Copyright © 2014 by the American Physiological Society.

35

Abstract 36 37 Hyperreflexia and spasticity are chronic complications in spinal cord injury (SCI), with 38

limited options for safe and effective treatment. A central mechanism in spasticity is 39

hyperexcitability of the spinal stretch reflex, which presents symptomatically as a 40

velocity-dependent increase in tonic stretch reflexes and exaggerated tendon jerks. 41

Here we test the hypothesis that dendritic spine remodeling within motor reflex 42

pathways in the spinal cord contributes to H-reflex dysfunction indicative of spasticity 43

after contusion SCI. Six-weeks after SCI in adult Spague-Dawley rats, we observed 44

changes in dendritic spine morphology on α-motor neurons below the level of injury, 45

including increased density, altered spine shape, and redistribution along dendritic 46

branches. These abnormal spine morphologies accompanied the loss of H-reflex rate-47

dependent depression (RDD) and increased H/M ratio. Above the level of injury, spine 48

density decreased compared with below-injury spine profiles and spine distributions 49

were similar to uninjured controls. As expected, there was no H-reflex hyperexcitability 50

above the level of injury in forelimb H-reflex testing. Treatment with NSC23766, a Rac1-51

specific inhibitor, decreased the presence of abnormal dendritic spine profiles below the 52

level of injury, restored RDD of the H-reflex, and decreased H/M ratios in SCI animals. 53

These findings provide evidence for a novel mechanistic relationship between abnormal 54

dendritic spine remodeling in the spinal cord motor system and reflex dysfunction in SCI. 55

56

57

58

59

Introduction 60

61

Hyperreflexia and spasticity, which arise in up to 60% of patients with spinal cord injury 62

(SCI), can severely affect quality of life, contribute to chronic pain, and lead to 63

musculoskeletal deformity (Skold et al., 1999; Walter et al., 2002). Although currently 64

available drugs, such as baclofen, can provide some relief, these drugs have limited 65

therapeutic utility and effectiveness. Thus, there is a significant need for a more 66

complete understanding of spasticity, and for more effective treatment after SCI. 67

68

Central mechanisms that underlie pathological reflex control after injury or disease 69

include the loss of cortical and local spinal inhibition, injury-induced plasticity, and 70

increased motor neuron excitability (Bennett et al., 2001a; Boulenguez et al., 2009; 71

Hultborn et al., 2007; Hunanyan et al., 2013). While plasticity between Ia afferents and 72

α-motor neurons shapes the H-reflex response in an activity-dependent manner in 73

human and rodent (Raisman, 1994; Thompson et al., 2009), maladaptive changes can 74

also contribute to pathological H-reflex function associated with spasticity—clinically 75

defined as a velocity-dependent increase in tonic stretch reflexes with exaggerated 76

tendon jerks, resulting from hyperexcitability of the spinal stretch reflex (Ashby et al., 77

1987; Lance, 1980; Nielsen et al., 2007). 78

79

Dendritic spines, micron-sized structures that are sites of postsynaptic activity, regulate 80

the efficacy of synaptic transmission and can thereby alter the electrical information 81

passing through circuit pathways (Bourne et al., 2007; Calabrese et al., 2006; Pongracz, 82

1985; Segev et al., 1988; Tan et al., 2009). Localized increases in synaptic strength 83

through the de novo formation and development of postsynaptic dendritic spines 84

constitute a persistent structural basis for learning and memory in the CNS (Xu et al., 85

2009; Yuste et al., 2001a). In the present study, we assess the possibility that 86

abnormalities in dendritic spine morphology on α-motor neurons contribute to the 87

persistent dysfunctional state within the spinal motor reflex pathway after SCI. 88

89

Our previous studies and evidence in the literature demonstrate that dendritic spine 90

morphology can change following disease or injury (Kim et al., 2006; Tan et al., 2012b; 91

Tan et al., 2013; Tan et al., 2008). Importantly, adverse changes in spine morphology 92

including (1) the elaboration from thin, filopodia-like spines to a mushroom shape, a 93

morphology associated with increased synaptic strength and stability (Yuste et al., 94

2001b); (2) an increase in spine density along dendrites, which provides more sites for 95

postsynaptic connections (Bonhoeffer et al., 2002), and a spatial redistribution of spines 96

along dendrites to locations closer to the cell body (Kim et al., 2006; Ruiz-Marcos et al., 97

1969) have been shown to contribute to neuronal hyperexcitability (Tan et al., 2009). 98

Although dendritic spine remodeling occurs in the motor cortex after SCI (Kim et al., 99

2006), no study has reported on dendritic spines located on spinal α-motor neurons. 100

Moreover, it is unknown whether SCI-induced changes in dendritic spine morphologies 101

can contribute to spasticity. 102

103

The activity of small GTP-binding protein Rac1 governs actin cytoskeleton 104

reorganization to regulate dendritic spine morphology (Tashiro et al., 2000; Tashiro et 105

al., 2004). Constitutively activated Rac1 increases the rate of dendritic spine turnover, 106

spine density and stability, and spine volume (Nakayama et al., 2000). In contrast, 107

dominant negative Rac1 expression or administration of a Rac1-specific inhibitor 108

NSC23766 disrupt dendritic spine formation and development (Tan et al., 2011; Tan et 109

al., 2012c; Tashiro et al., 2000; Tolias et al., 2007). Importantly, SCI increases Rac1 110

mRNA expression, with levels that can remain elevated for up to three months or more 111

(Dubreuil et al., 2003; Erschbamer et al., 2005). It is not known, however, if Rac1 112

activity contributes to dendritic spine remodeling and reflex dysfunction following SCI. 113

114

Here we provide the first evidence of dendritic spine plasticity on α-motor neurons after 115

SCI and demonstrate a structure-function relationship between dendritic spine 116

dysgenesis and exaggerated spinal motor reflexes associated with spasticity. Six weeks 117

after contusion SCI, animals exhibited increased H-reflex responsiveness (i.e., shown 118

by reduced rate-dependent depression or RDD). Histological assessment in these 119

animals demonstrated that α-motor neurons located below the level of injury within the 120

L4-L5 spinal segments had an increase in dendritic spine density, a significant 121

redistribution of spines along dendrites, and increased dendritic spine head-diameter—122

morphological profiles consistent with those shown to contribute to increased neuronal 123

excitability (Pongracz, 1985; Tan et al., 2009). In contrast, above the level of injury, we 124

observed an absence of exaggerated H-reflex response, reduced dendritic spine 125

densities, a close-to-normal distribution of spines, and normal dendritic spine length and 126

head diameter. Inhibition of Rac1 disrupted SCI-induced dendritic spine profiles on α-127

motor neurons below the injury site, reduced VGluT1 expression (a marker for 128

excitatory primary afferent terminals), and decreased H-reflex responsiveness. Taken 129

together, these observations provide evidence for a new perspective into mechanisms 130

of neuroplasticity within the spinal reflex pathway, and demonstrate a relationship 131

between dendritic spine remodeling and reflex dysfunction after SCI. Targeting of 132

molecular-pathways that regulate spine structure could represent a novel avenue for 133

managing spasticity after SCI. 134

135

Materials and Methods 136

137

Animals and spinal cord injury. Experiments were performed in accordance with the 138

National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All 139

animal protocols were approved by the Yale University Institutional Animal Use 140

Committee. Animals were housed under a 12 h light/dark cycle in a pathogen-free area 141

with water and food provided ad libitum. A total of 32 adult male Sprague-Dawley rats 142

(175-200 g; Harlan, Indianapolis, IN) underwent procedures to produce each treatment 143

group (Fig. 1, Study Design: Sham n = 11; SCI + Vehicle n = 11; SCI + anti-Rac n = 144

10). Animals were first divided into two treatment arms (Fig. 1). The first group received 145

a contusive spinal cord injury at the 2nd lumbar spinal segment (L2): animals were 146

anesthetized with a mixture of ketamine and xylazine (80/5 mg/kg, i.p.). A small 147

laminectomy was carefully performed at the 12th thoracic vertebra (T12), which exposed 148

the dorsal L2 spinal cord surface (Hebel, 1976). We stabilized the spinal cord in an 149

Infinite Horizon (IH) Impactor device (Precision Systems and Instrumentation [PSI], 150

Lexington KY) by clamping the rostral T11 and caudal T13 vertebral bodies with Adson 151

stabilizing forceps attached to the IH stage (Scheff et al., 2003). The spinal contusion 152

injury was performed with a metal rod (tip diameter of 2.5mm) that was applied to the 153

spinal cord surface with an impact force of 170 kdyn (Rabchevsky et al., 2003; Scheff et 154

al., 2003) (data shown in Fig. 2). For Sham animals (without SCI), the same surgical 155

procedure was followed; including placement of the animal within the IH stabilizing 156

forceps, except no contusion injury was performed. Following all surgical procedures, 157

muscle, fascia, and skin were sutured in sequential layers with 4-0 monofilament 158

sutures. Postoperative treatments included twice daily injections of 0.9% saline solution 159

for rehydration (3.0 cc/s.c.) and Baytril (0.3cc; 3.5 mg/kg b.w., sc, twice daily for 3 days) 160

to prevent urinary tract infection. 161

162

Behavior. Two experimenters blinded to group assignment evaluated animals using the 163

Basso, Beattie, Bresnahan (BBB) locomotor rating scale (Basso et al., 1995) for 164

validation of injury equivalency across SCI animals, as well as to determine whether 165

treatments had an effect on overall locomotor ability,. The BBB score (1 worst to 21 166

best) consists of a combination of hind-limb movements, trunk position and stability, 167

hind limb stepping and coordination, paw placement, and tail position. Behavioral 168

testing was performed at three time-points (Fig. 1): 1) on naïve animals prior to any 169

surgical procedures, 2) within one-week after catheter implantation (and before any 170

drug infusions at ~5-weeks post-SCI), and 3) immediately prior to experimental endpoint 171

(6-weeks post-SCI and Sham surgeries). Prior to any testing, animals were allowed to 172

acclimatize to the testing area for 60-90 minutes. During an experimental trial, animals 173

were allowed to roam freely in the test field (enclosed 3’ x 3’ flat surface) and a similar 174

four minute timeframe of movement was assessed by the experimenters using the BBB 175

scale, as previously described (Basso et al., 1995). After each trial, the surface was 176

cleaned with soap and water and dried. For analysis, BBB scores from both right and 177

left sides of animals were averaged and data from the two experimenters were 178

averaged within groups, and then statistically compared across groups. 179

180

Intrathecal catheter implantation and drug delivery. Five weeks after SCI or Sham 181

surgeries, all animals received ketamine/xylazine anesthesia (80/5 mg/kg, i.p.). As 182

described previously (Tan et al., 2012b), a small craniotomy was performed to expose 183

the atlanto-occiptal membrane (between the occipital bone and vertebral column 184

C1/atlas). A sterile 32G catheter (Recath Co., PA) was carefully inserted through a slit 185

in the membrane and threaded intrathecally until the tip of the catheter reached the 186

lumbar enlargement. The catheter was secured near the base of the skull with sutures 187

placed through overlying muscle and skin. To prevent leakage and infection, the 188

exposed rostral tip of the catheter was heat-sealed by pinching the end with a 189

sufficiently heated and sterilized forceps. The location of the caudal end of the catheter 190

was validated at the experimental endpoint after animals were sacrificed. Animals were 191

allowed to recover for 2-3 days after catheter implantation and then received one of two 192

infusions through the catheter: i) drug vehicle (0.9% sterile saline, 10μl volume, twice 193

daily for 3 days) and ii) NSC23766, a target-specific Rac1-GTPase inhibitor (EMD 194

Chemicals, Darmstadt, Germany), at 2.65 μg/μl (5μl volume, twice daily for 3 days) 195

followed by a 5μl sterile 0.9% saline flush. To measure the maximal affect of treatments, 196

experimental assessments were performed within 1-2 days following the last infusion of 197

vehicle or drug solution. We did not infuse NSC23766 drug in Sham animals in this 198

study, since we had previously already established that NSC23766 does not 199

significantly affect higher-order electrophysiological or behavioral function in uninjured, 200

control animals (Tan et al., 2012b). At the end of the study, this study design produced 201

four comparator arms (Fig. 1, gray): Sham, SCI + Vehicle (includes SCI (above) and 202

SCI (below)), and SCI + anti-Rac. 203

204

Histology. For Golgi-cox staining using a commercial kit and according to 205

manufacturer’s instructions (FD Neurotechnologies; Ellicot, MD) (Sham, n = 5; SCI + 206

Vehicle, n = 4; SCI + anti-Rac, n = 5), a subpopulation of rats from terminal 207

electrophysiological recordings (see below) under ketamine/xylazine anesthesia were 208

killed and processed. Spinal cord tissue (from the cervical enlargement, C4-C5, and 209

lumbar enlargement, L4-L5) was quickly removed (<5 minutes), rinsed in distilled water, 210

and immersed in the kit’s impregnation solution. After the incubation period (~3 weeks), 211

200μm-thick sections were cut on a vibratome (DTK-1000 microslicer; Ted Pella) and 212

mounted on gelatinized glass slides. Sections were stained, rinsed in distilled water, 213

dehydrated, cleared, and coverslipped with Permount medium. For 214

immunohistochemistry, remaining rats were deeply anesthetized with ketamine-xylazine 215

and transcardially perfused with 250 ml of 0.1M phosphate buffer (PB) at 37°C followed 216

by 300 ml of freshly prepared cold paraformaldehyde solution (4% in 0.1M PB). The 217

spinal cord was removed and post-fixed for 2 hours at room temperature, cryoprotected 218

by immersion in 30% sucrose, 0.1M PB at 4°C. Frozen coronal sections from C4-C5, 219

the injury site at L2, and L4-L5, were cut at 20μm thickness using a cryostat (Leica; 220

Bannockburn, IL). Sections were collected onto Superfrost plus slides (Fischer 221

Scientific; Pittsburgh, PA). Immunofluorescence staining methods are described 222

previously (Tan et al., 2006). Sections were washed in blocking solution (0.1 M PBS, 223

0.1% Triton X-100, and 4% normal donkey serum) and incubated overnight at 4°C in 224

mouse anti-VGlut antibody (University of California at Davis/NIH NeuroMab facility 225

1:1000), rabbit anti-GFAP (1:2000 Abcam) or rabbit anti- PKC-γ (Santa Cruz 226

Biotechnology 1:1000). After washing in blocking solution, sections were incubated in 227

fluorescent secondary antibodies, CY3 donkey anti-mouse (Jackson ImmunoResearch 228

Laboratories 1:500) or Alexa Fluor 488 donkey anti-rabbit (Invitrogen 1:2000). Sections 229

were visualized and digitally imaged using a Nikon Eclipse 80i fluorescence microscope 230

equipped with an HQ Coolsnap camera (Roper Scientific; Tucson, Arizona) or Nikon D-231

Eclipse C1 confocal microscopy system. Multi-capture mosaic images were digitally 232

stitched with NIS Elements software (Nikon Instruments, Inc.). 233

234

Dendritic spine visualization on motor neurons and analysis. Investigators blinded 235

to treatment conditions performed all imaging studies and analyses. To visualize 236

neurons and ultra-fine processes, we used a Golgi-staining method as previously 237

described (Tan et al., 2008). For our purpose, we required the ability to fully reconstruct 238

neuronal structure, which required that tissue be exposed to high-intensity light for long 239

periods of time (up for 4 hours per imaging session), which can quickly bleach or 240

diminish other visualization tools, e.g., fluorophores. Golgi staining permits the 241

identification and sampling of a relatively large number of neurons from cervical and 242

lumbar levels within the same animal, and provides robust visualization of the entire 243

neuronal structure, including detailed resolution of dendritic spines. We were specifically 244

interested in motor neuron pools that innervated muscle groups in the forelimb, i.e., 245

extensor carpi radialis, and hindlimb, i.e., plantar muscle. To identify these α-motor 246

neurons, we followed a screening workflow based on data from our previous study (Tan 247

et al., 2012a) and those previously validated in rats (Crockett et al., 1987; Hashizume et 248

al., 1988; Jacob, 1998). We began with a broad sample population of neurons by 249

identifying Golgi-stained α-motor neurons located in the ventral spinal cord in Rexed 250

lamina IX and with soma diameters >25μm (Hashizume et al., 1988; Jacob, 1998). 251

Above the injury site (C4-C5), we narrowed candidate neurons for analysis by selecting 252

neurons from motor pools located in similar dorsolateral coordinates of α-motor neurons 253

shown to innervate the extensor carpi radialis muscle group (~1.5-2 mm deep; 1.5-2 254

mm lateral from midline), as we and others have demonstrated through intramuscular 255

retrograde tracing studies (Sunshine et al., 2013; Tan et al., 2012a; Tosolini et al., 2013). 256

Below the injury site (L4-L5), we narrowed our sampled α-motor neurons to those 257

located in ventral motor pools with similar dorsolateral coordinates of motor pools 258

known to innervate the plantar muscle (~1.5-2.5 mm deep; 1.5 mm-2.2 mm lateral from 259

midline) as shown by retrograde tracing (Crockett et al., 1987; Jacob, 1998). As a 260

refinement step for analysis a priori, we only included α-motor neurons for analysis that 261

had 1) dendrites and dendritic spines that were clearly and completely impregnated, 2) 262

neurons that had dendritic branches appearing as a continuous length for at least 350 263

μm within the tissue slice, and 3) with at least one-half of the primary dendritic branches 264

that remained within the thickness of the tissue section, such that their endings were not 265

cut and appeared to taper into a complete ending (see representative neuron in Fig. 3). 266

To determine if there were any morphological differences across our sample neurons, 267

we used Neuroexplorer software (MicroBrightfield, Williston, VT) to measure maximum 268

cell diameter, aspect ratio (feret max/feret min), form factor (4π x area/perimeter2), 269

number of primary dendrites, and the total dendritic branch length of each treatment 270

arm and compared these morphometry values across treatment groups (Table 1). To 271

refine our identification and measurements of dendritic spines, specific morphological 272

characteristics were used (Kim et al., 2006; Tan et al., 2012b): we defined a spine neck 273

as the structure between the base of the spine and the interface with the parent dendrite 274

branch, and the base of the spine head where the appearance of the spine swells 275

distally into a bulb-like structure. Thin- and mushroom-shaped spines were classified as 276

follows: thin spines had head diameters that were less than, or equal to the length of the 277

spine neck, whereas mushroom spines had head diameters that were greater than the 278

length of the spine neck. These criteria for two spine geometric categories were used 279

because classification into only two spine shapes allowed us to use simple, but very 280

strict rules in classifying spine morphology. Although this approach prevented the 281

discrimination of subtle variations in spine shape, it allowed the collection of a very large 282

sample size and others and we have described the physiological characteristics of thin 283

and mushroom spine shapes on neuronal and circuit function (Bourne et al., 2007; 284

Holmes, 1990; Tan et al., 2009). Note that these criteria do not imply the physiological 285

characterization of the neurons we analyzed, but rather control for morphological 286

diversity within the sampled spinal motor neuron population (Kitzman, 2005; Tashiro et 287

al., 2003). 288

289

To digitally reconstruct motor neurons, we used a Neurolucida software suite (version 290

9.0; MicroBrightfield, Williston, VT) and a pen tablet (Intuos 5 touch, Wacom). We 291

analyzed the completed three-dimensional reconstructions of motor neurons for spine 292

density and distribution. Each imaging session consisted of a contour map (outline of 293

the spinal cord section with location of identified neuron) and the motor neuron, which 294

was traced in the X-, Y-, and Z-axis. Dendritic spine type were located and marked on 295

each reconstructed dendritic branch (thin spines, blue; and mushroom spines, red). 296

297

length. To determine any changes in spatial distribution of dendritic spines relative to 298

the cell body, we used a Sholl’s analysis (Tan et al., 2008). 299

bins were formed around each cell body and spine density within each bin was 300

averaged within each treatment group. For statistical comparison, spine density at 301

dendrite branch locations within 50-150 μm (proximal bins) and 200- μm (distal 302

bins) from the cell body were pooled and compared across treatment groups. 303

304

To determine changes in spine dimensions, five neurons were arbitrarily chosen from 305

each treatment group and visible spines were measured for spine length and spine 306

head diameter (Kim et al., 2006). Spine length was defined as the distance from the tip 307

of the spine to the junction of the spine to the main dendrite branch. Spine head 308

diameter was defined as the longest line drawn normal to the length of the parent 309

dendrite branch. A total of 411 dendrites from 82 identified α-motor neurons (in 4-5 310

animals per treatment group) were included in our analyses (Sham = 118; SCI (below 311

injury) = 116; SCI (above injury) = 90; SCI + anti-Rac = 87). 312

313

Areal density of VGluT1 labeling. To examine changes in the number of VGluT1-314

expressing synaptic terminals, we assessed areal density of VGluT1 expression using a 315

modified approach described previously (Tan et al., 2012a). High-resolution digital 316

photographs were taken at 10x magnification and combined into a single mosaic image 317

of the entire spinal cord at L4-L5. We photographed 10 sections per animal. Sections for 318

analysis were chosen on the basis of tissue integrity (e.g., no major tears) and 319

equivalent loss of PKC-γ immunoreactivity in the dorsal CST of the spinal cord dorsal 320

columns. Sections were aligned according to the point of intersection between the gray 321

matter above the central canal and the dorsal median septum. All images underwent 322

threshold adjustments using equivalent contrast/brightness levels to highlight only 323

VGluT1 expressing puncta (Photoshop, Adobe, San Jose, CA). Images were binarized 324

and color-inverted for analysis. For color-coded heat maps in Fig. 9, binarized images 325

were exported into MATLAB (Mathworks, Natick, MA) and averaged using custom 326

scripts, as described previously (Brus-Ramer et al., 2007; Friel et al., 2007). For image 327

analysis, mosaic images of each spinal cord coronal section were divided into three 328

dorso-ventral regions corresponding to the dorsal zone (~lamina I-III), the intermediate 329

zone (~lamina IV-VI), and the remaining ventral horn of the gray matter (Tan et al., 330

2012a). Because we were only interested in VGluT1 in the gray matter, the white matter 331

areas were digitally removed prior to analyses. Because VGluT1 expressing puncta 332

were visually distinct from each other, without overlap, the number of VGluT1 puncta 333

were easily counted in each region using ImageJ software 334

(http://rsb.info.nih.gov/ij/index.html). Data from gray matter regions were pooled within 335

groups and compared across experimental treatment groups. 336

337

H-reflex testing. Terminal electrophysiological experiments were performed 6 weeks 338

after SCI or Sham surgeries. Because H-reflex responses in rats under ketamine 339

anesthesia resemble those seen in unanesthetized humans (Ho et al., 2002) and does 340

not alter the time course of presynaptic inhibition, which may occur with other 341

anesthetics (e.g., sodium pentobarbital) (Tang et al., 1973), we anesthetized animals 342

with an induction dose of ketamine and xylazine (80/5mg/kg, i.p.) and maintained on 343

ketamine alone (20 mg/kg, i.p.) (Ho et al., 2002; Hosoido et al., 2009). Core body 344

temperature was monitored with a rectal thermometer and maintained at 38 ± 1°C with 345

a circulating water heating pad placed under an absorbent pad. To record 346

electromyogram (EMG) data, which included the muscle response (M wave) and the 347

monosynaptic reflex response (H reflex) above and below the injury site in SCI animals, 348

we used an established percutaneous needle preparation (Boulenguez et al., 2009; Lee 349

et al., 2009; Schieppati, 1987; Thompson et al., 1992a; Valero-Cabre et al., 2004). We 350

chose to use this minimally invasive procedure because it is analogous to methods 351

used to evoke and record H-reflex in humans (Palmieri et al., 2004; Schieppati, 1987) 352

and provides the opportunity to stimulate and record at all four limbs within the same 353

animal without perturbing muscle or nerve tissue that would otherwise be disrupted from 354

more invasive surgical electrode placement, e.g., cuff electrode implants. In addition, 355

this recording approach maintains the integrity of the vascular system and optimizes the 356

tissue preservation and collection methods for histological study (see above) that we 357

performed following electrophysiological experiments. For stimulation, a pair of Teflon 358

insulated stainless steel fine wire electrodes (0.002” bare metal diameter; A-M Systems, 359

Inc., Carlsborg, WA) were threaded into a 32 G syringe needle. The wire ends were 360

carefully bent into sharp barbs, the insulation was removed with heat (to expose tips 361

~1mm), and the needle and wire was then transcutaneously inserted until the wire tip 362

was in close proximity to the mixed nerves of the deep radial nerve or tibial nerve, 363

above or below the injury site in SCI animals, respectively. The needle was retracted 364

and the wire remained in place. The second electrode was inserted similarly, spaced 365

~2mm apart from the first electrode. Stimulating electrode placement was adjusted until 366

the intensity of square wave stimulating pulses (0.2 ms duration continuously given at a 367

rate of 1 every 3 seconds) required to induce subtle visible motor twitch responses, i.e., 368

wrist extension/radial abduction or plantar flexion was below 1mA (Lee et al., 2009; 369

Valero-Cabre et al., 2004). For recording electrodes, we used insulated wire electrodes 370

made of similar materials and exposed ~2 mm of the wire tips using heat. To record 371

EMG data from the forelimb (e.g., brachioradialis reflex), an electrode was inserted into 372

the interosseous muscles between the fourth and fifth digit and a reference electrode 373

was placed subcutaneously in the dorsolateral surface of the paw. To record EMG data 374

from the hindlimb (e.g., plantar reflex), an electrode was inserted into the plantar 375

muscles within the palmar/ventral surface of the hindpaw and proximal to the ankle 376

region. A reference electrode was placed subcutaneously within the dorsolateral surface 377

of the hindpaw. These reflexes were chosen based on our pilot experiments and 378

previous work that demonstrated EMG reflex response evoked in these muscles could 379

be reproducibly recorded after SCI (Boulenguez et al., 2010; Kim et al., 2009; Valero-380

Cabre et al., 2004). Note that SCI-induced changes in the plantar reflex, primarily 381

innervated by motor pools in L5, less from L4 (Crockett et al., 1987), have been shown 382

to be similar to changes in reflexes elicited in other hindlimb muscles, i.e., tibialis 383

anterior and gastrocnemius, which are also innervated by L4 and L5 (Lee et al., 2009; 384

Valero-Cabre et al., 2004). EMG responses were filtered (10-1000 hz), amplified, and 385

recorded for offline analysis using Spike 2 (version 7.08; CED Software, Cambridge, 386

England). To identify optimal stimulation intensity for activating stable M-wave and H-387

reflex responses, square wave pulses (0.2 ms duration) were applied at a rate of 1 388

every 3 seconds. The intensity of electrical stimulation was first adjusted to determine 389

the minimum intensity to evoke an M-wave response ~50% of the time and 390

progressively increased until a stable M-wave and maximal H-reflex response could be 391

observed. 392

393

To measure rate-dependent depression (RDD) of the H-reflex response, we performed 394

a paired-pulse stimulation paradigm with a conditioning and test pulse that we applied at 395

a range of interpulse intervals (10 ms to 2000 ms). Three trials (10 sweeps/trial) with at 396

least 30 seconds between trials were recorded for each interpulse interval. The M and H 397

response wave amplitudes were quantified from averaged and rectified waveforms 398

within each animal (Boulenguez et al., 2010; Tan et al., 2012a). For comparison across 399

treatment groups, the maximum waveform amplitudes of the H and M response to the 400

test pulse were converted into a percentage of the maximum amplitude response to the 401

conditioning pulse. M and H waveform amplitudes were measured from baseline to 402

peak amplitude. To determine trial-to-trial consistency, the coefficient of variation (CoV) 403

from the M and H waves were calculated by dividing the standard deviation with the 404

mean maximum amplitude. Following recording experiments, animals were sacrificed 405

for Golgi-staining or immunohistological study as described above. 406

407

A number of studies have shown that dendritic spine morphology can change within 408

minutes following cortical injury or activity (Majewska et al., 2003; Mizrahi et al., 2004; 409

Zhang et al., 2005). Moreover, others have shown that exogenous electrical stimulation 410

can induce plasticity of the H-reflex, which can persistent for many hours or days (Chen 411

et al., 2003; Chen et al., 2006). Although in this study our H-reflex testing was 412

performed acutely (i.e., lasting ~1 hour per animal), we ensured that all animals, 413

including both spinal cord injured and uninjured, Sham animals, underwent similar H-414

reflex testing protocols to control for potential confounds of direct nerve stimulation. 415

416

Statistical analysis. All statistical tests were performed at the alpha-level of 417

significance of 0.05 by two-tailed analyses using parametric or non-parametric tests, as 418

appropriate. For comparisons of anatomical and functional changes above and below 419

the injury site after SCI, we compared multiple comparisons of data collected within the 420

same animals and with Sham animals. To determine the appropriate statistical model to 421

apply to these specific datasets, we incorporated two assumptions: 1) that the above 422

and below datasets were dependent variables affected by the application of the 423

independent treatment variable, SCI, and 2) as a consequence of SCI, a putative 424

secondary pathway interaction arises between above and below injury spinal segments 425

that can affect the above or below datasets (e.g., an emergent interaction following SCI 426

that leads to differential affects on spinal cord tissue located above or below the SCI 427

injury site). Given these assumptions, our datasets fit most closely with a one-way (or 428

one-factor) ANOVA statistical model, where SCI is the treatment factor applied to two 429

dependent variables, above and below. Moreover, to control for multiple comparison 430

errors with the additional comparison against the Sham group, we applied a post-hoc 431

repeated measures correction. While we considered the application of a split-plot 432

analytical model, this approach falsely assumes that the secondary, post-SCI interaction 433

is a factor that is independent of the first-cause treatment factor, SCI. In summary, for 434

comparing datasets gathered from above and below the injury site, we applied 435

repeated-measures corrections (Dunn or Bonferonni post hoc tests) following ANOVA 436

and Kruskal-Wallis one-way ANOVA on ranks analyses. As a note, previous reports 437

have similarly applied ANOVA repeated measures design to compare ipsilateral and 438

contralateral sides of the spinal cord following unilateral nerve trauma, or compare 439

regions above and below the injury site after SCI. The statistical design of these studies 440

encompassed the assumed effect of a post-injury secondary, bidirectional interaction, 441

(Anderson et al., 1998; Chang et al., 2010; Tal et al., 1994; Tan et al., 2012a; Tan et al., 442

2007), which may have also emerged within our experimental SCI model. Data 443

management and statistical analyses were performed using SigmaPlot (version 12.5; 444

Systat Software Inc.) and Microsoft Office Excel (2011). Data in the text are described 445

as mean ± SD. All graphs are plotted as mean ± SEM using SigmaPlot. 446

447

448

449

Results 450

451

Contusion SCI disrupts the dorsal corticospinal tract 452

In SCI animals, contusion injury at spinal cord segment L2 resulted in severe damage of 453

the dorsal columns and gray matter (Fig. 2A), in contrast with Sham (Fig. 2B). 454

Histological examination of caudal spinal cord tissue (segments below L3) showed no 455

visible tissue damage or glial scar tissue (not shown). The IH Impactor device provided 456

biomechanical measurements during each SCI procedure (Fig. 2C and Fig. 2D). The 457

cord surface displacements upon rod impact were not significantly different across both 458

SCI groups, SCI + Veh and SCI + anti-Rac (Fig. 2C; 1419.8 ± 293.3 vs. 1394.9 ± 266.2 459

μm, p>0.05, t test). Similarly, there was no difference in the actual applied force 460

between the SCI groups (Fig. 2D; 172.4 ± 12.4 vs. 181.4 ± 19.7 kdyn, p>0.05, ANOVA 461

on ranks with Dunn’s post hoc). Applied impact force with the IH device predicts the 462

amount of tissue sparing, which correlates closely with locomotor functional outcome 463

(Scheff et al., 2003). PKC-γ immunoreactivity served as an anatomical marker to help 464

confirm the injury magnitude of our SCI model (Bradbury et al., 2002; Sasaki et al., 465

2009; Tan et al., 2012a). In coronal spinal cord sections above the injury in the cervical 466

enlargement, PKC-γ immunoreactivity symmetrically labeled the dorsal corticospinal 467

tract (dCST) and small-diameter cells located in laminae I/II (Fig. 2E) (Mori et al., 1990). 468

Six weeks after SCI, below the injury level in the lumbar enlargement, PKC-γ staining of 469

the dCST was bilaterally eliminated from the dorsal columns (Fig. 2F). PKC-γ staining 470

profiles of the dCST and superficial laminae remained intact (data not shown) in cervical 471

and lumbar enlargement tissues in Sham animals. 472

473

Dendritic spine density changes on motor neurons after SCI 474

Dendritic spines remodel in the motor cortex after SCI (Kim et al., 2008; Kim et al., 475

2006); however, it is not known whether SCI-induced dendritic spine dysgenesis occurs 476

on α-motor neurons within the spinal cord. Injury-induced changes in dendritic spine 477

morphology on nociceptive neurons in the dorsal horn have been shown to contribute to 478

increased excitability associated with neuropathic pain (Tan et al., 2009; Tan et al., 479

2012b; Tan et al., 2008). To determine whether dendritic spine remodeling occurs on 480

spinal cord α-motor neurons, we identified α-motor neurons (see Materials and 481

Methods) and performed a morphological comparison of α-motor neurons across 482

treatment groups (Fig. 3). We identified α-motor neurons located in lamina IX and within 483

motor pools in the lateral regions of the ventral horn (Fig. 3A and Fig. 3B). Six-weeks 484

after SCI, motor neurons had widely projecting dendritic trees containing numerous 485

spines. Qualitative observations demonstrated marked differences in spine number 486

across treatment arms (Fig. 3C-F). To ensure equivalent sampling across groups, we 487

assessed several morphological criteria and compared these values across treatment 488

groups (Table 1). There were no statistically significant differences in maximum cell 489

diameter, aspect ratio, form factor, number of primary dendrites, or total dendrite branch 490

lengths (for all comparisons: p>0.05). We therefore interpreted any differences in 491

dendritic spine profiles across groups as not due to variations in neuronal sampling and 492

rather an effect of experimental treatments. As a note, the values for maximum cell 493

diameter, form factor, and dendritic branch lengths were similar to measurements for α-494

motor neurons that were labeled by intramuscular injected retrograde tracers (Bose et 495

al., 2005; Crockett et al., 1987; Hashizume et al., 1988; Jacob, 1998). 496

497

To obtain an accurate measure of dendritic spine profiles from ventral spinal cord tissue, 498

we digitally reconstructed α-motor neurons using Neurolucida software (Fig. 4). We 499

marked the location of sample neurons on a contour map of the spinal cord gray matter 500

(Tan et al., 2008). α-motor neurons from each treatment group (Fig. 4A-D; red dots; 501

n=20-21 cells/group) were located in the ventrolateral regions of the gray matter, shown 502

as a single representative contour from segmental level C5 (above injury) or L5 (below 503

injury). Dendritic spines on traced motor neurons were marked along dendritic branches 504

and color-coded with thin-shaped (blue) or mushroom-shaped (red) spines (Fig. 4A’-D’). 505

506

A main objective of this study was to assess the contribution of SCI-induced changes in 507

dendritic spines to reflex dysfunction; we therefore measured three morphological 508

profiles of spines that have been associated with injury-induced neuronal 509

hyperexcitability: 1) increased density of dendritic spines, particularly mature 510

mushroom-spine spines, 2) redistribution of spines toward dendritic branch locations 511

close to the cell body, and 3) enlargement of the spine head diameter. Because 512

spasticity often presents below the injury site following SCI, and less commonly above 513

(Skold et al., 1999), we measured dendritic spines on α-motor neurons in motor pools of 514

the cervical (C4-C5; above injury) and lumbar (L4-L5; below injury) spinal segments that 515

innervate forelimb and hindlimb musculature, respectively (see Methods and Materials). 516

517

As shown in Fig. 5A, six weeks after SCI, total dendritic spine density on motor neurons 518

below the injury site increased compared with Sham control and neurons above the 519

injury site (p<0.05; 2.80 ± 0.78 vs. 1.82 ± 0.52 vs. 1.10 ± 0.54 spines/10 μm dendrite; 520

ANOVA on ranks with Dunn’s post hoc). In contrast, motor neurons above the injury site 521

in the cervical enlargement had dendritic spine densities that decreased compared with 522

Sham (p<0.05). A similar profile of dendritic spine density was also observed for thin-523

shaped dendritic spines (Fig. 5B): below the injury site there was a significant increase 524

in thin spines compared with above the injury and Sham Sham (p<0.05; 2.42 ± 0.63 vs. 525

1.03 ± 0.54 vs. 1.61 ± 0.40 spines/10 μm dendrite; ANOVA on ranks with Dunn’s post 526

hoc). Importantly, there was a significant increase in the density of mature, mushroom-527

shaped spines located on α-motor neurons below the injury site compared with Sham 528

and above the injury (p<0.05; 0.45 ± 0.33 vs. 0.20 ± 0.26 vs. 0.06 ± 0.06 spines/10 μm 529

dendrite; ANOVA on ranks with Dunn’s post hoc) (Fig. 5C). Note that the mushroom-530

spine density observed below the injury site after SCI represents a more than 200-700% 531

increase compared with mature-shaped spine densities above the injury site and Sham 532

control. 533

534

Dendritic spines redistribute toward proximal branches on motor neurons after 535

SCI 536

Excitatory afferent inputs located closer to the neuronal cell body can have a greater 537

weighted impact upon the overall electrical output of a neuron due to the closer 538

proximity to the axon hillock (Pongracz, 1985; Tan et al., 2009; Yuste et al., 2004). To 539

profile changes in dendritic spine distribution along motor neuron branch processes, we 540

applied a Sholl’s analysis and pooled spine densities within proximal regions close to 541

the cell body (50-150 μm) and distal regions (200-350 μm) (see Methods and Materials) 542

(Fig. 5D-F). 543

544

On motor neurons below the injury site in SCI animals, total spines, thin-shaped, and 545

mushroom-shaped dendritic spines increased on proximal dendrite branches compared 546

with equivalent regions in Sham and SCI neurons above the injury site (p<0.05; for total 547

spines: 3.1 ± 1.1 vs. 2.1 ± 1.1 vs. 1.2 ± 0.69; for thin spines: 2.7 ± 0.16 vs. 1.9 ± 0.05 vs. 548

1.1 ± 0.05; for mushroom spines: 0.39 ± 0.04 vs. 0.17 ± 0.06 vs. 0.08 ± 0.01 spines/10 549

μm dendrite; one-way ANOVA with Bonferroni’s post hoc) (Fig. 5D-F). At distal regions, 550

SCI did not change spine density of any category on motor neurons from below the 551

injury compared with Sham (p>0.05). On the other hand, motor neurons below the injury 552

had increased spine density for all categories as compared with above the injury at 553

distal regions (p<0.05; for total spines: 3.2 ± 1.4 vs. 1.4 ± 0.9; for thin spines: 2.3 ± 1.4 554

vs. 1.4 ± 0.9; for mushroom spines: 0.85 ± 0.9 vs. 0.08 ± 0.16 spines/10 μm dendrite; 555

ANOVA on ranks with Dunn’s post hoc). There was no difference in any spine densities 556

at distal regions on neurons from Sham compared with neurons above the injury in SCI 557

animals (p>0.05). 558

559

Dendritic spine dimensions change on motor neurons after SCI 560

To quantify the effects of SCI on spine length and spine head diameter, we analyzed 561

880 to 1,305 dendritic spines that were sampled from 4-7 motor neurons per group (see 562

Materials and Methods; Sham n = 3 animals/5 neurons; above SCI level n = 3 animals/7 563

neurons; below SCI level n = 3 animals/4 neurons). As shown in Fig. 5G, dendritic 564

spines below the injury site in SCI animals decreased in length compared with above 565

the injury site and Sham (p<0.05; 1.38 ± 0.79 μm vs. 1.67 ± 0.97 μm vs. 1.60 ± 0.81 μm, 566

one-way ANOVA with Bonferroni’s post hoc). In contrast, spine-head diameter 567

increased after SCI below the injury site compared with above the injury and Sham 568

(p<0.05; 1.22 ± 0.65 μm vs. 0.97 ± 0.57 μm and 0.95 ± 0.56 μm, one-way ANOVA with 569

Bonferroni’s post hoc) (Fig. 5H). Motor neurons above the injury site after SCI did not 570

differ compared with Sham in any dimension measured (p>0.05). 571

572

H-reflex response increases below the injury site after SCI 573

Dendritic spine morphology significantly influences synaptic function, i.e., in a structure-574

function relationship (Pongracz, 1985; Segev et al., 1988; Segev et al., 1998). As we 575

and others have shown (Leuner et al., 2004; Majewska et al., 2000; Tan et al., 2009; 576

Zhou et al., 2004), increased dendritic spine density, mature dendritic spine 577

morphologies, e.g., mushroom-shapes, and proximal redistribution of spine synapses 578

can amplify neuronal excitability, enhance frequency-following ability, reduce noise-579

filtering capabilities, and attenuate inhibitory input. To determine the effect of SCI on 580

reflex function in association with dendritic spine remodeling on α-motor neurons, we 581

measured the H-reflex response in uninjured Sham, i.e., in the hindlimb, and SCI 582

animals above and below the injury in the forelimb and hindlimb, respectively. 583

We also measured the M-wave, which reveals the electrical responsiveness of motor 584

axons, its ability to conduct an action potential, and the electrochemical coupling of the 585

efferent and muscle tissue, e.g., neuromuscular junction (Hultborn et al., 1995). In 586

normal animals, the H-reflex undergoes activity rate-dependent depression (RDD). A 587

reduction in H-reflex RDD is a physiological indicator of spasticity (Boulenguez et al., 588

2010; Ho et al., 2002; Lee et al., 2009; Taylor et al., 1984). To determine H-reflex and 589

M-wave response in SCI animals, we electrically stimulated the deep radial nerve or 590

tibial nerves and recorded reflex response from muscle in the forelimb, extensor carpi 591

radialis, and hindlimb, plantar muscle. As a comparison, we measured evoked H- and 592

M-response from hindlimb muscle in uninjured Sham animals. We chose these reflexes 593

based on preliminary studies and previously published work that demonstrated that 594

evoked reflex responses for these muscles could be reproducibly produced in adult rats 595

after SCI (Boulenguez et al., 2010; Kim et al., 2009; Valero-Cabre et al., 2004). 596

Importantly, previous studies have shown that changes in plantar reflex after SCI are 597

similar to changes in reflexes elicited for other hindlimb muscles, i.e., tibialis anterior 598

and gastrocnemius, which are also innervated by motor pools in L4/L5 (Lee et al., 2009; 599

Valero-Cabre et al., 2004). 600

601

We used a paired-pulse stimulation paradigm: a control and test pulse, separated by a 602

range of interpulse intervals from 2000 ms to 10 ms (Fig. 6). A representative trace in 603

Sham produced from recordings of plantar muscle shows two evoked EMG waves: the 604

M-response and the H-reflex (central loop pathway) (Fig. 6A). As the interval between 605

the control and test pulse shortened from 2000 ms to 10 ms, there was a marked 606

depression of the H-reflex. The M-wave amplitude also decreased with shortening 607

interpulse intervals, demonstrating rate-dependent depression of motor neuron-to-608

muscle response. Fig. 6B and Fig. 6C shows a qualitative example of SCI-induced 609

reductions in H- and M-wave RDD from muscle recordings above and below the injury 610

at 100ms and 10ms interpulse intervals. In SCI animals, the M-wave appeared to 611

maintain stable amplitude even at shorter interpulse intervals. 612

613

We quantified the percentage change in H-reflex for uninjured Sham (n = 7) and SCI 614

animals (n = 6) over the range of interpulse intervals (Fig. 6D). In Sham animals, the H-615

reflex maintained stable amplitude between 2000ms and 300 ms and with a steady 616

decline at shorter interpulse intervals, similar to that observed in previous studies (Ho et 617

al., 2002; Hosoido et al., 2009; Tan et al., 2012a). The M-wave amplitude in Sham 618

animals remained stable through a wider range of interpulse intervals, 2000 ms and 100 619

ms (%M-wave amplitude 2000 vs. 50ms: p<0.05, one-way ANOVA with Bonferroni’s 620

post hoc) (Fig. 6E). Therefore, the H-reflex depression response in sham animals is not 621

due to the inability of muscle to respond to repeated stimulus activity. At shorter 622

interpulse intervals (i.e., 3-5 ms), both the H-reflex and M-wave responses depressed at 623

a much greater rate and, in most stimulus-recording trials, failed to appear in sufficient 624

number for analysis (data not shown). 625

626

Six-weeks after SCI, H-reflex measurements from evoked hindlimb reflex revealed a 627

significant reduction in RDD, i.e., H-reflex amplitude stabilized, through the entire range 628

of interpulse intervals tested (Fig. 6D). Between interpulse intervals 50 to 150 ms, there 629

was a significant increase in H-reflex response in SCI below the injury site compared 630

with Sham (p<0.05; one-way ANOVA with Bonferroni’s post hoc). Notably, SCI 631

appeared to amplify the reflex response below the injury site at the shortest interpulse 632

interval at 10 ms compared with Sham (p<0.05; one-way ANOVA with Bonferroni’s post 633

hoc), with amplitude responses greater than 100% of control amplitude. In contrast, 634

within the same SCI animal, there continued to be significant RDD in recordings above 635

the injury at 500 ms, 50 ms, and 10 ms (p<0.05; one-way ANOVA with Bonferroni’s post 636

hoc) (Fig. 6D). H-reflex RDD above the injury was not significantly different to Sham 637

across all interpulse intervals (p > 0.05). These findings indicate that SCI-induced 638

increases in H-reflex response only occurred below the level of the injury and from 639

muscle innervated primarily by motor pools in spinal segment L4-L5. Over the range of 640

interpulse intervals tested from 2000 ms to 100 ms, the %M-wave amplitude remained 641

close to 100% across all comparator groups (Fig. 6E). However, there was a significant 642

increase in %M-wave amplitude above 100% of control in SCI above or below injury 643

compared with Sham, which depressed at 50ms and 10ms (p<0.05; ANOVA on ranks 644

with Dunn’s post hoc). The H/M ratio calculated from reflex data in SCI below the injury 645

was larger than Sham or above injury at all interpulse intervals (Fig. 6F). SCI below the 646

injury site resulted in a more stabilized rate of decay for H/M ratio values, an indication 647

of hyperreflexia and spasticity (Little et al., 1985; Matthews, 1966; Nielsen et al., 2007). 648

649

To assess changes to H-reflex response fidelity, we calculated the coefficient of 650

variation (CoV = StDev/mean of %H-reflex amplitude) for 50 ms and 10 ms for Sham 651

and SCI animals. At the 50 ms interpulse interval, the CoV after SCI below the injury 652

was nearly 30-50% smaller than for SCI above the injury or Sham (SCI below injury, 653

0.20; SCI above injury, 0.58; Sham, 0.85). Similarly, the CoV for SCI below the injury 654

site was almost 40-60% smaller than Sham (SCI below injury, 0.37; SCI above injury, 655

0.66; Sham, 0.92). Taken together, these values show that in addition to increasing H-656

reflex amplitude, SCI also increases the reliability of reflex activation below the injury 657

site. 658

659

Inhibition of Rac1 disrupts dendritic spine remodeling 660

We reasoned that if abnormal dendritic spine profiles after SCI contributes to increased 661

reflex excitability, then disruption of dendritic spine remodeling would reduce signs of 662

spasticity. To determine whether disruption of dendritic spine remodeling on α-motor 663

neurons in L4-L5 after SCI reduces spasticity, we assessed the effects of administering 664

NSC23766, a specific Rac1-GTPase inhibitor. Treatment with NSC23766 resulted in a 665

decrease in total, thin-, and mushroom-shaped spine density compared with SCI + Veh 666

(p<0.05, SCI + anti-Rac vs. SCI + Veh; 0.95 ± 0.24 vs. 2.8 ± 0.78 total spines/10 μm 667

dendrite; 0.92 ± 0.23 vs. 2.4 ± 0.63 thin spines/10 μm dendrite; 0.03 ± 0.03 vs. 0.44 ± 668

0.33 mushroom spines/10 μm dendrite, ANOVA on ranks with Dunn’s post hoc) (Fig. 669

7A-C). We also determined the effect of NSC23766 treatment on dendritic spine 670

distribution by measuring spine density in proximal and distal locations along dendrites 671

of α-motor neurons after SCI (Fig. 7D-F). NSC23766 treatment significantly decreased 672

spine density in proximal and distal regions and for all spine categories, including total 673

spines, thin-shaped, and mushroom-shaped dendritic spines (p<0.05; for proximal total 674

spines: 3.1 ± 0.2 vs. 0.9 ± 0.4; for proximal thin spines: 2.7 ± 0.16 vs. 0.9 ± 0.4; for 675

proximal mushroom spines: 0.4 ± 0.4 vs. 0.02 ± 0.02; for distal total spines: 3.2 ± 0.5 vs. 676

1.2 ± 0.5; for distal thin spines: 2.4 ± 0.5 vs. 1.1 ± 0.5; for distal mushroom spines: 0.8 ± 677

0.9 vs. 0.04 ± 0.07 spines/10 μm dendrite; one-way ANOVA with Bonferroni’s post hoc). 678

679

Dendritic spines on α-motor neurons in SCI animals that were treated with NSC23766 680

decreased in spine length and head diameter compared with SCI + Veh (p<0.05; length, 681

0.7 ± 0.42 μm vs. 1.38 ± 0.79 μm; head diameter, 0.93 ± 0.51 μm vs. 1.23 ± 0.66 μm, 682

ANOVA on ranks with Dunn’s post hoc) (Fig. 7G-H). Together, these findings show that 683

Rac1-inhibitor NSC23766 treatment can effectively disrupt SCI-induced dendritic spine 684

remodeling on α-motor neurons. 685

686

Inhibition of dendritic spine remodeling reduces H-reflex excitability after SCI 687

Previous work has demonstrated that intrathecal infusion of NSC23766 is efficacious in 688

restoring close-to-normal dendritic spine profiles on nociceptive neurons within the 689

dorsal horn after SCI and peripheral nerve injury (Tan et al., 2011; Tan et al., 2008). In 690

these studies, treatment with NSC23766 also reduced neuronal hyperexcitability 691

associated with central sensitization, demonstrating that Rac1-regulated dendritic spine 692

remodeling can contribute to mechanisms underlying neuropathic pain (Tan et al., 693

2012c). To determine whether disruption of Rac1-regulated dendritic spine profiles on α-694

motor neurons attenuates exaggerated H-reflex responsiveness after SCI, we used a 695

paired-pulse stimulation paradigm as described above (Hultborn et al., 1995; Tan et al., 696

2012a) (see Fig. 6). Representative EMG traces in SCI animals below the injury in the 697

hindlimb demonstrates that stimulation produced both M-wave and H-reflex responses 698

(Fig. 8). At the shortest interpulse interval at 10 ms, there was a notable reduction of the 699

RDD. At the 10 ms interpulse interval, treatment with the Rac1-inhibitor NSC23766 700

appeared to restore RDD of the H-reflex in hindlimb EMG recordings (e.g., reduced H-701

reflex amplitude in response to the test pulse) (Fig. 8B). 702

703

Fig. 8C shows the quantified changes in the H-reflex response in SCI + Vehicle (n = 6) 704

and SCI + anti-Rac1 (n = 5). Six weeks after SCI, in animals treated with control vehicle, 705

the hindlimb H-reflex response demonstrated almost no RDD, with %H-reflex response 706

remaining stable (e.g., close to 100%) across the range of interpulse intervals from 707

2000 ms to 10 ms (for a comparison against Sham, see Fig. 6). Treatment of SCI 708

animals with the Rac1-inhibitor resulted in a restoration of RDD at the three shortest 709

interpulse intervals at 100 ms, 50 ms, and 10 ms compared with SCI + Vehicle (p<0.05; 710

at 100 ms, 56.9 ± 29.9% vs. 91.6 ± 8.5%; at 50 ms, 34.5 ± 35.3% vs. 92.4 ± 19.3%; at 711

10 ms, 13.3 ± 21.4% vs. 88.9 ± 32.9%, one-way ANOVA with Bonferroni’s post hoc) 712

(Fig. 8C). In comparisons across SCI animal groups, the %M-wave amplitude remained 713

close to 100% between interpulse intervals 2000 ms and 100 ms (group means: for SCI 714

+ Veh, 98.6 ± 1.5%; for SCI + anti-Rac1, 107.3 ± 11.4%) (Fig. 8E). The %M-wave 715

response at shorter interpulse intervals, 50 ms and 10 ms, exhibited greater variability 716

compared with longer stimulus intervals, but was not statistically significant (p > 0.05). 717

718

We calculated the coefficient of variation of the %H-reflex at 50ms and 10ms across 719

SCI animal groups treated with vehicle or the Rac1-inhibitor. At the 50ms and 10ms 720

interpulse intervals, treatment with the Rac1-inhibitor in SCI animals resulted in a CoV 721

that was nearly 4-fold greater than vehicle treated SCI animals (SCI + Veh, 0.27 – 0.37; 722

SCI + anti-Rac, 0.9 – 1.6). Thus, in addition to restoring RDD, Rac1-inhibition also 723

increased the variability of the reflex response. Fig. 8E shows the plot for the H/M ratio. 724

Treatment with the Rac1-inhibitor decreased the overall H/M ratio across all interpulse 725

intervals tested between 2000 ms and 10 ms, as shown by a downward shift in trend 726

line slope. 727

728

VGluT1 bouton areal density in the gray matter does not increase after injury 729

Synapse-associated protein markers (i.e., synaptophysin and PSD-95) increase after 730

SCI, demonstrating the presence of injury-induced synaptic plasticity (Tan et al., 2008; 731

Tan et al., 2012c). Because upper motor tract injury and SCI can increase the 732

excitability of spinal reflex pathways below the injury (Baastrup et al., 2010; Little et al., 733

1985; Tan et al., 2012a), we next determined if excitatory inputs, particularly those of IA 734

afferents, change after SCI. Vesicular glutamate transporter-1 (VGluT1) is a widely used 735

marker for excitatory Ia afferent terminations in the spinal cord (Alvarez et al., 2011; 736

Alvarez et al., 2004; Kitzman, 2007). 737

738

As shown in representative images in Fig. 9A-D, immunopositive VGluT1 puncta were 739

distributed throughout the spinal cord gray matter of the lumbar enlargement, L4-L5, for 740

each analyzed treatment group (Fig. 9A-D, left panels). To visualize the distribution of 741

VGluT1 puncta in the gray matter, we compiled the VGluT1-staining profiles from 742

multiple tissue sections from each treatment group and produced spatial heat maps (Fig. 743

9A-C, right panels). Although the highest concentration of VGluT1 positive boutons 744

appeared to correspond with Rexed laminae 5-6 (Hantman et al., 2010; LaMotte et al., 745

1991), VGluT1 puncta were distributed throughout all laminae. The areal densities of 746

VGluT1 boutons were calculated in the total gray matter (Fig. 9D), and within three 747

regions: the dorsal horn, the intermediate zone, and the ventral horn (Fig. 9E-G; also 748

see insets). Six weeks after SCI, we observed no significant difference in the areal 749

density of VGLUT boutons in the total gray matter compared with uninjured Sham 750

animals (p>0.05). Similarly, there was no statistical difference following SCI + Veh in the 751

other three gray matter regions analyzed, as compared with uninjured Sham (p>0.05). 752

In contrast, treatment with NSC23766 significantly decreased the areal density of 753

VGluT1 compared with SCI + Veh in the total gray matter, in the intermediate zone, and 754

the ventral horn (for total gray matter: 63.6 ± 37.1 vs. 124.5 ± 57.7; for intermediate 755

zone: 72.4 ± 25.2 vs. 138.7 ± 51.3; for ventral horn: 58.1 ± 48.4 vs. 129.8 ± 55.9 puncta; 756

ANOVA on ranks with Dunn’s post hoc). There was no significant change in areal 757

density of VGluT1 in the superficial dorsal horn following Rac1-inhibitor treatment in SCI 758

animals as compared with SCI + Veh (p > 0.05). 759

760

Rac1 inhibition does not affect locomotor behavior 761

To rule out any differences in gross locomotor ability in SCI animals, we assessed post-762

injury locomotor behavior using the Basso, Beattie, and Bresnahan (BBB) locomotor 763

scale (Basso et al., 1995; Basso et al., 1996) (Fig. 10). Blinded observers performed 764

behavioral testing at three time-points: on naïve animals prior to any surgical 765

procedures, within one-week after catheter implantation and before drug treatment, and 766

at the six week post-SCI endpoint (also see Fig. 1). All naive animals exhibited a 767

baseline locomotor score of 21 (1 worst to 21 best). Five weeks after SCI and catheter 768

implantation, before any treatments, animals exhibited a mean BBB score of 13.6 ± 3.8, 769

demonstrating the expected locomotor ability in the late SCI recovery phase (Basso et 770

al., 1995). BBB testing of SCI animals after vehicle or drug delivery demonstrated no 771

significant affect on locomotor ability with scores remaining unchanged between SCI 772

animals treated with (n = 10) or without Rac1 inhibitor (n = 11) (p >0.05, 14.2 ± 1.6 vs. 773

13.8 ± 2; ANOVA on ranks). 774

775

Discussion 776

777

Spinal cord circuits can reorganize, changing in structure and function after injury 778

(Raisman, 1991). Our present findings demonstrate robust changes in dendritic spine 779

morphology on α-motor neurons after SCI, including an increase in dendritic spine 780

density, a distribution of spines closer to the cell body, and the presence of more mature 781

dendritic spines. These postsynaptic dendritic changes have been shown to accompany 782

increased neuronal excitability after SCI (Rall et al., 1992; Segev et al., 1998; Tan et al., 783

2009). In agreement, we observed a significant loss of H-reflex RDD below the injury 784

(i.e., increased H/M ratio), indicative of spasticity (Boulenguez et al., 2010; Matthews, 785

1966; Nielsen et al., 2007). Importantly, dendritic spines above the injury exhibited a 786

nearly opposite morphological profile with decreased spine density, and distribution and 787

shape that were more similar to control. As expected, there was no change in H-reflex 788

excitability above the level of injury. Overall, these results demonstrate that abnormal 789

dendritic spine profiles below the level of injury accompany spasticity after SCI, and 790

conversely, the lack of such spine profiles above the injury correspond with a lack of 791

spinal reflex hyperexcitability. 792

793

To further elucidate the structure-function link between dendritic spine dysgenesis and 794

hyperreflexia, we disrupted dendritic spine remodeling by targeting Rac1-signaling in 795

SCI animals. We have previously shown that Rac1 inhibition disrupts dendritic spine 796

remodeling in dorsal horn sensory neuron after SCI, nerve injury, and diabetes mellitus 797

(Tan et al., 2011; Tan et al., 2012b; Tan et al., 2008). In the present study, we observed 798

a decrease in spine density on α-motor neurons and a closer-to-normal distribution of 799

dendritic spines following treatment with NSC23766, a Rac1-inhibitor. NSC23766 800

treatment also decreased spine length and head diameter, and partially restored normal 801

H-reflex activity (i.e., increased RDD). Taken together, our study is the first to 802

demonstrate robust dendritic spine reorganization on α-motor neurons in the ventral 803

horn, which accompanies spasticity after SCI. We implicate Rac1-signaling as an 804

important mediator in both the structural and functional changes within the spinal reflex 805

pathway after injury. 806

807

Spasticity after SCI has been attributed to a variety of mechanisms within the spinal 808

reflex arc (Nielsen et al., 2007; Roy et al., 2012). Muscle spindle afferents may lose 809

either presynaptic inhibition or reciprocal inhibition. Alternatively, loss of Renshaw 810

interneuron activity, thought to mediate reciprocal inhibition, can trigger spasticity 811

(Nielsen et al., 2007). Evidence obtained from intracellular recordings of spinal 812

motoneurons also demonstrates increased motoneuron excitability, i.e., the ability to 813

generate action potentials, including the appearance of plateau potentials and persistent 814

inward sodium and calcium currents in rat motor neurons after SCI (Bennett et al., 815

2001b; Heckmann et al., 2005; Li et al., 2004), KCC2 downregulation (Boulenguez et al., 816

2010; Vinay et al., 2008), and sodium channel misexpression (Harvey et al., 2006; Li et 817

al., 2003). Inflammation, e.g., microgliosis, occurs in a number of nervous system injury 818

models, including SCI (Craner et al., 2005; Hains et al., 2006), and may contribute to 819

increasing excitability of neuronal populations within spinal circuits (Gwak et al., 2009; 820

Zhao et al., 2007b). Astrocyte activation after injury can potentially maintain 821

hyperexcitability (Scholz et al., 2007). Finally, maladaptive plasticity such as ‘collateral’ 822

or ‘reactive’ sprouting may contribute to altered spinal motor control (Boulenguez et al., 823

2010; Krenz et al., 1998; Nielsen et al., 2007; Raisman, 1994). Others have shown 824

altered dendrite branch length on motor neurons in the spinal cord that accompanies 825

spasticity after SCI (Kitzman, 2005). Although dendritic spine morphologies change on 826

pyramidal neurons in the motor cortex after SCI (Kim et al., 2006), the functional role for 827

these spine alterations is not firmly understood. Computer simulations have attempted 828

to predict the physiological contribution of dendritic spines on motor neurons (Rall et al., 829

1967); however, in vivo changes in dendritic spine structure have not been reported in 830

spinal cord motor pools. 831

832

Dendritic spine morphology partly determines synaptic function, and therefore provides 833

a visual clue into how neural networks function (Calabrese et al., 2006; Segev et al., 834

1998). Dendritic spines can reorganize rapidly following synaptic activity, e.g., activity-835

dependent plasticity, and increase in density which provides new or stronger synapses 836

(Halpain, 2000). Abnormal dendritic spine morphologies have been reported in a wide 837

spectrum of neuropsychiatric diseases, including post-traumatic stress disorder, 838

substance dependence and addiction, autism spectrum disorders, and mental 839

retardation (Halpain et al., 2005; Purpura, 1974). Although adaptive plasticity between 840

Ia afferents and spinal motor neurons can shape H-reflex response in both humans and 841

rodents (Thompson et al., 2009; Wolpaw, 1994), maladaptive plasticity can contribute to 842

pathological H-reflex function associated with hyperreflexia and spasticity (Lance, 1980; 843

Nielsen et al., 2007). In chronic SCI, hyperexcitability of the spinal stretch reflex, e.g., H-844

reflex, is thought to underlie spasticity, which manifests as a velocity-dependent 845

increase in tonic stretch reflexes, with uncontrollable “jerking” movement and abnormal 846

muscle tone, whereby muscle continually contract (Ashby et al., 1987; Lance, 1980; 847

Nielsen et al., 2007; Skold et al., 1999). In our study, we observed significant SCI-848

induced changes in dendritic spine morphologies on α-motor neurons below the injury 849

site, which accompanied a loss of RDD and a stabilization of the H/M ratio over a broad 850

range of nerve stimulation rates. Importantly, we observed only minor changes in M-851

wave response after SCI, indicating that changes in RDD and H/M ratio were primarily 852

due to mechanistic changes within the spinal cord monosynaptic circuit. 853

854

Although sacrocaudal injuries might better replicate some aspects of clinical spasticity 855

(Li et al., 2003; Ritz et al., 1992), these SCI models only allow studies of neurological 856

deficits in tail musculature, which are absent in human. To permit sufficient locomotor 857

ability for open-field behavioral assessment and H-reflex testing of hindlimb musculature, 858

a parallel of leg muscle groups in human, we performed contusion SCI at spinal 859

segment L2. As with all SCI animal studies, however, we encountered an observation 860

suggesting that our injury model also cannot entirely reflect the human SCI condition. In 861

contrast with human SCI at lower-thoracic or upper lumbar segments, which generally 862

produces some chronic negative motor signs, including flaccidity or lower limb 863

weakness (Doherty et al., 2002), we observed increased spinal motor reflex activity 864

associated with increase muscle tone six weeks after injury. Thus, it is important to 865

mention that our contusion SCI model was utilized as a compromise to study spinal 866

reflex function in hindlimb musculature. 867

We noted in our study that the RDD of the H-reflex did not exhibit depression at a 868

similar rate compared with an earlier report of the effect of spinal contusion on RDD 869

(Thompson et al., 1992b). Whereas Thompson et al. observed activity-rate depression 870

of approximately 85% of control at 5 Hz (Thompson et al., 1992a); we observed a 871

similar loss of H-reflex activity at 10-20 Hz (i.e., 50-100ms interpulse interval). A 872

probable explanation for this discrepancy is due to the additional procedures that 873

animals underwent prior to reflex testing in our study, including surgical implantation of 874

intrathecal catheters and infusions of vehicle or drug solutions. Although no effect of 875

catheter implantation and drug infusion have been observed in previous nociceptive 876

testing in control animals (Tan et al., 2008), it is possible that these additional 877

experimental procedures could have led to sensitization of afferents within the spinal 878

reflex circuit. Nonetheless, the magnitude of activity-rate depression in Sham and SCI 879

animals in our study fell within other documented ranges (Hosoido et al., 2009; Tan et 880

al., 2012a). 881

882

Exogenous electrical stimulation of the H-reflex circuit can directly induce changes in H-883

reflex response, which can persist for many hours or days (Chen et al., 2003; Chen et 884

al., 2006). These previous studies demonstrate the presence of activity-dependent 885

plasticity within the monosynaptic reflex system. In our current study, to establish a 886

structure-function relationship between dendritic spine remodeling and H-reflex 887

dysfunction after SCI, we were required to assess spine changes and H-reflex function 888

within the same animals. This acute H-reflex function and dendritic spine assessment 889

approach limited our ability to control for the possible confound that dendritic spine 890

changes could have also resulted from EMG reflex testing, which required direct 891

stimulation of muscle-sensory nerves. Although H-reflex testing only lasted about an 892

hour per animal and we ensured that all animals underwent similar testing protocols, we 893

cannot exclude the possibility that each treatment group could have had a different 894

capacity to respond to potential EMG testing-induced dendritic spine changes. We note 895

that in uninjured Sham animals, there were a greater proportion of thin-shaped dendritic 896

spines compared with mushroom-shaped dendritic spines (see Fig. 4 and 5). Thin, 897

filopodia-like dendritic spines are thought to represent newly formed or more plastic 898

dendritic spines; whereas mushroom shaped spines may represent more stable, mature 899

spines (Bourne et al., 2007; Bourne et al., 2008). This observation in Sham animals 900

does suggest the possibility that EMG testing could have resulted in the de novo 901

presence of more structurally responsive thin-shaped dendritic spines. Our current 902

experiments, however, do not permit us to determine whether spine changes are a sole 903

result of treatments, i.e., SCI, drug intervention, etc., or a combination of treatments and 904

the potential direct affect of EMG electrophysiological assessment, which could have 905

also influenced spine morphologies. 906

907

Our findings raise the question of how altered dendritic spine morphologies below the 908

level of injury contribute to hyperexcitable spinal reflex function. The dendritic spine 909

profiles we observed after SCI below the injury can have direct biophysical effects on 910

motor neuron excitability (Rall et al., 1967; Segev et al., 1988; Tan et al., 2009). The 911

distribution of spine synapses closer to motor neuron cell bodies can increase the 912

overall impact of excitatory input and transmission. The increased head volume of 913

mature, mushroom-shaped dendritic spines permits increased clustering of excitatory 914

membrane receptors, i.e., AMPA receptors (Wiens et al., 2005). These properties can 915

amplify synaptic transduction. Importantly, we observed an increase in mature spine 916

geometries after SCI, which could increase input discretization (e.g., narrow EPSP 917

waveforms) and allow trains of suprathreshold potentials to propagate at higher rates of 918

activity with greater fidelity (Rall, 1967; Rall et al., 1967). This transmission property 919

could contribute to excessive firing in motor neurons (Tan et al., 2009; Yuste et al., 920

2004) and facilitate supra- and sub-threshold temporal summation (Carter et al., 2007; 921

Gold et al., 1994; Martiel et al., 1994). In our study, temporal summation would most 922

likely have occurred at the shorter interpulse intervals during H-reflex testing. 923

Consecutive transcutaneous electrical stimulations to facilitate withdrawal reflex have 924

demonstrated maximal temporal facilitation at the 10-20 Hz range (Arendt-Nielsen et al., 925

1997; Arendt-Nielsen et al., 2000), suggesting the presence of a temporally dependent 926

integration mechanism within reflex pathways. Thus, it is interesting to consider that 927

SCI-induced dendritic spine changes leads to an amplification of presynaptic excitatory 928

input and enhancement of a temporally dependent integration mechanism to increase 929

the gain of H-reflex function after SCI. 930

931

We have implicated Rac1 in the pathological changes that contribute to chronic 932

nociceptive hyperexcitability after SCI (Tan et al., 2008). Rac1 is a small molecule 933

GTPase that is involved in regulating dendritic spine morphology (Tashiro et al., 2008). 934

In a mouse model of Fragile X syndrome (FMR knockout), Rac1 dysfunction is 935

associated with abnormal dendritic spine morphology and decreased pain (Chen et al., 936

2010; Lee et al., 2003; Price et al., 2007). In our model of SCI, we administered 937

NSC23766, a Rac1-specific inhibitor that does not affect cdc42 or Rho GTPases, or 938

affect the interaction of Rac1 to its downstream effector PAK1 (Gao et al., 2004). 939

Although our current study does not preclude off-target effects, we have previously 940

shown that the Rac1 inhibitor disrupts the development and appearance of dendritic 941

spines in vitro (Tan et al., 2011) and that NSC23766 treatment is effective in attenuating 942

dendritic spine remodeling in the dorsal horn after SCI (Tan et al., 2008). Moreover, 943

NSC23766 treatment does not significantly affect electrophysiological or behavioral 944

signs of pain-reflex withdrawal function in normal, uninjured animals (Tan et al., 2012b). 945

Although microglial activation has been implicated in altering the excitability of sensory 946

neurons after injury (Tan et al., 2012a; Zhao et al., 2007a), administration of NSC23766 947

does not appear to affect the activation of microglia after nerve injury (Tan et al., 2011). 948

In agreement with previous studies (Beauparlant et al., 2013; Kitzman, 2007), we report 949

that VGluT1 expression did not change after SCI, suggesting that the presence of H-950

reflex hyperexcitability is not due to increased excitatory presynaptic afferent inputs 951

(Kitzman, 2007). Although it is possible that Rac1-inhibition may have affected 952

presynaptic elements, NSC23766 treatment only decreased VGluT1 areal density in the 953

intermediate zone and ventral horn. This suggests that the Rac1-inhibitor had a 954

topographically specific effect on deeper lamina only, where sensory-motor neuron 955

populations exhibit dendritic spine plasticity after injury (Tan et al., 2011; Tan et al., 956

2008). 957

958

A major challenge in SCI research has been identifying the mechanisms that contribute 959

to secondary complications such as spasticity and pain. Our previous work has 960

demonstrated that dendritic spine remodeling in the dorsal horn contributes to 961

hyperexcitability associated with neuropathic pain after SCI (Tan et al., 2008; Tan et al., 962

2012c). Here we show that dendritic spine dysgenesis on α-motor neurons also 963

contribute to the development of spasticity. Our data further show that Rac1 signaling 964

participates in this dendritic spine remodeling and may provide a novel opportunity to 965

specifically address the underlying pathophysiology of spasticity after SCI. 966

967

968

969

Figure Legends 970

971

Fig. 1. Study design. All weight-matched animals underwent BBB locomotor testing to 972

obtain baseline behavioral data. Animals (n values) in each group are shown. In week 1, 973

animals were randomly assigned to receive Sham or SCI surgical procedures. In week 974

5, animals received intrathecal catheter implants. After 2-3 days of recovery, we 975

performed pre-treatment BBB testing and immediately administered intrathecal 976

infusions of control vehicle or NSC23766 (twice a day for 3 days). At experimental 977

endpoint at week 6, these treatments produced four comparator groups (gray shade). 978

Note that within SCI animals treated with vehicle we assessed and compared data 979

outcomes from above or below the injury site, i.e., forelimb and hindlimb. At endpoint, 980

we also performed post-treatment BBB testing, H-reflex assessment, and histological 981

analysis. 982

983

Fig. 2. Spinal cord injury. (A) Contusion injury at L2 resulted in severe damage of the 984

dorsal columns and gray matter, as shown by GFAP staining in coronal spinal cord 985

tissue sections. Asterisks (*) denotes lesion epicenter. (B) Intact spinal cord tissue from 986

Sham. (C-D) Biomechanical data provided by the IH impactor demonstrated no 987

difference between vehicle and Rac1-inhibitor treated SCI groups. (E) Six weeks after 988

SCI, PKC-γ) staining produced bilateral labeling of the dorsal corticospinal tract (dCST) 989

and lamina I/II. (F) At the lumbar level, L5, (below the injury), the absence of PKC-γ 990

immunoreactivity in the dorsal columns white matter tracts demonstrated significant 991

disruption of the dCST. SCI did not affect PKC-γ staining in superficial laminae. Scale 992

bars = 500 μm 993

994

Fig. 3. Golgi staining of spinal cord tissue reveals dendritic spines on motor 995

neurons in the ventral gray matter. (A) Image of the ventral gray matter with an 996

identified α-motor neuron located in Rexed lamina IX (arrow and white box). (B) High 997

power field of motor neuron shown in the inset in panel A. Six weeks after Sham and 998

SCI, representative images of dendritic branches show apparent differences in dendritic 999

spine profiles from (C) Sham, (D) SCI + Vehicle above the injury, (E) SCI + Vehicle 1000

below the injury, and (F) SCI with NSC23766, Rac1-inhibitor, treatment groups. (C’, D’, 1001

E’, F’) High magnification of selected dendrite regions from panels C-F (red boxes). 1002

Scale bar for A = 500 μm; B = 100 μm; C-F = 10 μm; C’-F’ = 2 μm 1003

1004

Fig. 4. Digital reconstructions of spinal cord motor neurons. To obtain an accurate 1005

profile of dendritic spines in motor neurons, we digitally reconstructed the entire branch 1006

structure of sampled neurons. (A, B, C, D) Contour traces from each group show the 1007

locations of all sampled motor neurons (red dots) within the gray matter (representative 1008

black trace). Density and distribution were measured from three-dimensional neuron 1009

reconstructions from (A’) Sham, (B’) SCI + Vehicle above the injury, (C’) SCI + Vehicle 1010

below the injury, and (D’) SCI + Rac1-inhibitor treatment. (A”, B”, C”, D”) An ~50 μm 1011

length of dendrite from neurons shown in panels A’-D’ (gray shaded region) show thin-1012

shaped (blue dots) and mushroom-shaped spines (red dots). Scale bar in A, B, C, D = 1013

500μm; A’, B’, C’, D’ = 50μm, A”, B”, C”, D” = 10μm 1014

1015

Fig. 5. Quantitative analysis of dendritic spine profiles between Sham and SCI 1016

animals above or below the injury. Analysis of dendritic spine profiles reveals 1017

differences in dendritic spine density (top row), distribution (middle row), and shape 1018

(bottom row). (A) Total dendritic spine density, which includes all spine shapes, (B) thin-1019

spines, and (C) mushroom spines decreased on motor neurons located above the injury 1020

after SCI compared with Sham (* = p<0.05). In contrast, total spine density increased on 1021

motor neurons located below the injury compared with either Sham or above the injury 1022

after SCI (* = p<0.05). Dendritic spine distribution for (D) total, (E) thin, and (F) 1023

mushroom spines differed across the comparator groups. At proximal regions in SCI 1024

animals, all spine densities increased below the injury as compared with Sham and 1025

motor neurons above the injury (* = p<0.05). In contrast, neurons above the injury had 1026

less total and thin-shaped spine density at proximal regions compared with Sham and 1027

below the injury (* = p<0.05). (F) Although mushroom-spine density on motor neurons 1028

above the injury did not differ from Sham at proximal regions, these neurons had 1029

significantly less mushroom spine density compared with below the injury. At distal 1030

regions, motor neurons below the injury had greater spine density in all categories 1031

compared with motor neurons above the injury (* = p<0.05). There was no difference in 1032

any spine densities at distal regions on neurons from Sham and above the injury in SCI 1033

animals. Dendritic spine shape analysis revealed no change in (G) spine length or (H) 1034

spine head diameter on motor neurons located above the injury in SCI animals 1035

compared with Sham (* = p<0.05). Below the injury, these measurements demonstrated 1036

a decrease in spine length, and an increase in spine head diameter compared with 1037

neurons in Sham and above the injury (* = p<0.05). 1038

1039

Fig. 6. Rate-dependent depression of the H- and M- responses above and below 1040

SCI. As a physiological assessment of the monosynaptic H-reflex, we performed a 1041

paired-pulse stimulation protocol. Representative traces (averaged 10-20 traces) of the 1042

M- and H-responses to control (first) and test (second) pulse in (A) Sham, (B) SCI 1043

above the injury, and (C) SCI below the injury. The control and test pulses were 1044

separated with a range of interpulse latencies between 2000 – 10ms. Note that in Sham 1045

animals, as the interpulse intervals decreased (e.g., increasing the rate of activity) 1046

between the test and control pulse, the amplitude of the M- and H-response decreased. 1047

As shown in panel C, in SCI below the injury, rate-dependent depression in amplitude of 1048

either the M- or H-response failed to appear. (D) %H-reflex and (E) %M-wave 1049

amplitudes are normalized values of the evoked stimulus-response of the test and 1050

control pulse. (D) After SCI, there was no significant difference in % H-reflex in SCI 1051

above the injury compared with Sham at any interpulse interval. In contrast, in SCI 1052

animals below the injury, the %H-reflex significantly increased compared with Sham at 1053

the shortest interpulse intervals between 100-10ms (* = p<0.05), demonstrating a loss 1054

of RDD and increased excitability of the H-reflex. Similarly, %H-reflex below the injury 1055

was significantly greater than above the injury in SCI animals at 500, 50, and 10ms 1056

interpulse intervals (§ = p<0.05). (E) % M-wave demonstrated a significantly increased 1057

response below the injury compared with both Sham (* = p<0.05) and above the injury 1058

(# = p<0.05) response. (F) The H/M ratio was calculated from M-wave and H-wave 1059

responses. 1060

1061

Fig. 7. Rac1-inhibitory treatment disrupts dendritic spine morphology on motor 1062

neurons in the ventral horn after SCI. Treatment with NSC23766 in SCI animals (SCI 1063

+ anti-Rac) resulted in a significant decrease in (A) total, (B) thin, and (C) mushroom-1064

spine density compared with SCI + Veh (* = p<0.05). (D, E, F) Assessment of dendritic 1065

spine distribution on motor neurons showed that NSC23766 treatment in SCI animals 1066

resulted in decreased spine density for all spine categories at both proximal and distal 1067

branch regions (* = p<0.05). NSC23766 treatment decreased SCI-induced (G) spine 1068

length and (H) spine head diameter compared with SCI + Veh (* = p<0.05). 1069

1070

Fig. 8. Disruption of Rac1-regulated dendritic spines reduces SCI-induced H-1071

reflex hyperexcitability. Representative traces show the M- and H-responses from 1072

paired-pulse testing in (A) SCI + Veh, below the injury and (B) SCI + anti-Rac treatment. 1073

(C) Quantification of the %H-reflex response demonstrated that the H-reflex response in 1074

SCI + Veh animals exhibited reduced RDD (also see Fig. 6). Rac1-inhibitor treatment in 1075

SCI animals reduced the %H-reflex at 100, 50, and 10 ms as compared with SCI + Veh 1076

(* = p<0.05). (D) There was no significant difference in the %M-wave between SCI + 1077

Veh and SCI + anti-Rac. (E) Treatment with the Rac1-inhibitor in SCI animals 1078

decreased the H/M ratio compared with SCI + Veh, as demonstrated by a steeper 1079

downward trend line. 1080

1081

Fig. 9. Excitatory terminals in the spinal cord gray matter. VGluT1-immunopositive 1082

puncta appeared throughout all laminae of the spinal cord gray matter in the lumbar 1083

enlargement, L4-L5 (left panels in A-C). Spatial heat maps (right panels in A-C; red 1084

highest density, blue lowest density) shows the overall areal density of VGluT1 1085

expression in (A) Sham, (B) SCI + Veh, and (C) SCI + anti-Rac treatment. 1086

Quantification of the VGluT1 puncta within the (D) total gray matter region, (E) the 1087

dorsal horn, (F) the intermediate zone, and (G) the ventral horn (panel insets and gray 1088

shade) demonstrated no significant change in SCI + Veh compared with Sham. 1089

Treatment with the Rac1-inhibitor in SCI animals decreased VGluT1 areal density 1090

compared with SCI + Veh in the total gray matter, intermediate zone, and ventral horn 1091

only (* = p<0.05) with no significant change in the dorsal horn (p > 0.05). The areal 1092

density of VGluT1 decreased in SCI + anti-Rac1 compared with Sham in the dorsal horn 1093

(* = p<0.05). Scale bar for A, B, C = 500 μm. 1094

1095

Fig. 10. Locomotor testing. Blinded observers performed BBB testing on animals at 1096

three time points: before any procedure, before treatment, and after treatment. All naive 1097

animals exhibited a baseline locomotor score of 21. There were no significant 1098

differences in BBB scores across group (p>0.05). 1099

1100

1101

1102

1103

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Acknowledgements: The work is supported by grants from the Paralyzed Veterans of 1445

America (PVA) and the Department of Veterans Affairs (VA) Medical Research Service 1446

and Rehabilitation Research Service. Andrew M. Tan is funded by the PVA Research 1447

Foundation and a VA Career Development Award (1 IK2 RX001123-01A2). The Center 1448

for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed 1449

Veterans of America with Yale University. We thank Pamela Zwinger and Peng Zhao for 1450

their excellent technical assistance. 1451

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Table 1. Spinal cord motor neuron morphometry

Maximum cell

diameter (um) Aspect Ratio Form Factor

Number of

primary

dendrites

Total dendrite

length (m)

Sham 54.6 ± 19.4 0.57 ± 0.17 0.49 ± 0.17 8.16 ± 3.69 1073.0 ± 671.4

SCI (above injury) 46.7 ± 11.7 0.51 ± 0.16 0.50 ± 0.16 5.73 ± 1.95 815.10 ± 451.9

SCI (below injury) 51.4 ± 17.4 0.64 ± 0.13 0.62 ± 0.09 4.80 ± 1.23 739.30 ± 280.6

SCI + anti-Rac 46.1 ± 8.7 0.63 ± 0.11 0.60 ± 0.10 4.38 ± 1.15 1085.8 ± 307.9 Data are shown as mean ± SD