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iScience Therapeutic Potential of 5HT2CR in Alleviating Spasticity and Pain via

Modulation of Motor Neurons and Interneurons in Spinal Cord Injury

Han Gong, Zuliyaer Talifu, Yu-Zhe Sun, Zhao-Ming Yue, Xiao-Xia Du, Liang-Jie Du

PII:

S2589-0042(25)01750-X

DOI:

https://doi.org/10.1016/j.isci.2025.113489

Reference:

ISCI 113489

To appear in:

iScience

Received Date: 19 November 2024
Revised Date: 15 June 2025
Accepted Date: 29 August 2025

Please cite this article as: Gong, H., Talifu, Z., Sun, Y.-Z., Yue, Z.-M., Du, X.-X., Du, L.-J., iScience

Therapeutic Potential of 5HT2CR in Alleviating Spasticity and Pain via Modulation of Motor Neurons and

Interneurons in Spinal Cord Injury

iScience

(2025), doi:

https://doi.org/10.1016/j.isci.2025.113489

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iScience

Therapeutic Potential of 5HT2CR in Alleviating Spasticity

and Pain via Modulation

of Motor Neurons and Interneurons

in Spinal Cord Injury

Han Gong

1,2,3,4,6

, Zuliyaer Talifu

5,6

, Yu-Zhe Sun

1,3

, Zhao-Ming Yue

1,3

, Xiao-Xia Du

1,4

, Liang-

Jie Du

1,3,7*

1 School of Rehabilitation, Capital Medical University, Beijing, China

2 School of Biological Science and Medical Engineering, Beihang University, Beijing

3 Department of Spinal and Neural Functional Reconstruction, China Rehabilitation Research

Center, Beijing, China

4 School of Rehabilitation Sciences and Engineering, University of Health and Rehabilitation

Sciences, Qingdao, China

5 School of Population Medicine and Public Health, Chinese Academy of Medical Sciences/Peking

Union Medical College, Beijing, China

6 These authors contributed equally

7 Lead contact

*Correspondence: duliangjie-ccmu@foxmail.com(L.J.D)

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SUMMARY

Numerous adverse outcomes can be resulted in limb spasm and spasticity that occur after spinal

cord injury (SCI). Here, we used adeno-associated virus (AAV) vectors and Cre-driver transgenic

mice to selectively overexpress 5-hydroxytryptamine2C receptor (5HT2CR) in motor, GABAergic,

and Glutamatergic interneurons. Targeted overexpression in these neuronal populations attenuated

spasticity-like responses and hindlimb motor deficits, and improved sensory abnormalities and

affective disturbances. These behavioral improvements were associated with changes in potassium

chloride cotransporter-2 (KCC2), gamma-aminobutyric acid type A receptor (GABAAR), and N-

methyl-D-aspartate receptor (NMDAR) expression in spinal cord tissue, indicating that the activity

of motor neurons is influenced by the expression of 5HT2CR. This work indicates that 5HT2CR

represents a promising target for alleviating spasticity and neuropathic pain after SCI.

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INTRODUCTION

Muscle spasticity is a common complication of spinal cord injury (SCI), representing a sensory-

motor control disorder following upper motor neuron damage, characterized by intermittent or

persistent involuntary muscle activation

. Approximately two-thirds of SCI patients experience

varying degrees of muscle spasticity within the first six months after injury. Spasticity can result in

stiffness of the limbs, sleep disturbances, spasmodic pain, and even spastic paralysis, significantly

impacting patients' daily care, rehabilitation training, and quality of life

2-4

. Treatment for spasticity

after SCI encompasses both conservative and surgical interventions

5-7

. While the current therapeutic

modalities lack curative efficacy and exhibit inherent deficiencies

, it is imperative to seek effective

relief and treatment methods for spasticity after SCI.

The integrity of the 5HT (5-hydroxytryptamine or serotonin) system in the spinal cord is crucial for

regulating sensation and coordinating motor functions

. Cental serotonin deficiency impairs

sensory-motor recovery after SCI

. The severity of spinal cord injury correlates positively with the

extent of damage to 5HT neuron axons,making the 5HT system a promising target for treating

muscle spasticity post-injury

11,12

. Various 5HT receptors, such as serotonin 1A

13,14

, 1B

,, 2A

, 2B

and 2C

, have been investigated for their therapeutic potential in addressing SCI and its

complications.Following SCI, disruption of 5HT projections results in reduced serotonin levels at

the injury site

. This depletion may lead to hypersensitivity or constitutive activation of specific

5HT receptors involved in spinal cord network regulation. To date, numerous strategies targeting

the 5HT system have shown potential in improving motor function and coordination in rodent

models of spinal cord injury. The 5HT system directly affects neuronal excitability, which depends

on the location and activation of 5HT receptors that modulate Na+, K+, and Ca2+ channel states

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Furthermore, the 5HT system regulates glutamatergic and GABAergic neurotransmissions,

indirectly influencing motor neuron excitability by acting on spinal interneurons

. Thus, enhancing

the 5HT system's function may alleviate sensory and motor deficits, reduce muscle spasms, and help

restore normal physiological functions following spinal cord injury.

The mechanisms of spasticity after spinal cord injury are complex and still debated. These

mechanisms includes:1. hyperactivity of fusiform muscle movement;2. reduction of post-activation

depression (PAD);3. reduction of presynaptic inhibition;4. reduction of Ia reciprocal inhibition;5.

increased excitability of motor neurons;6. sustained activation of persistent inward currents (PICs);7.

differential regulation of spinal cord neurons by descending drive from monoamine

neurotransmitters;7. dysregulation of excitation/inhibition (E/I) input in interneurons

. However, the

direct cause of spasticity is the excessive excitability of motor neurons. Therefore, it is an urgent

challenge to balance excitatory and inhibitory signals, regulate motor neuron activity, and suppress

the occurrence and progression of limb spasticity, all while not hindering the recovery of motor

function after spinal cord injury.

The regulation of the descending monoaminergic system may be a crucial breakthrough for

achieving the aforementioned goals. Part of the cause of muscle spasticity following spinal cord

injury is associated with the loss of the brainstem-derived serotonergic system. It has been found

that 5HT2CR can regulate motor neuron excitability by modulating persistent inward currents

(PICs). Although this modulation can enhance muscle contraction function lost after SCI, it also

contributes to increased spasticity

In our previous studies, a finding that piqued our interest. we used adeno-associated virus (AAV) to

overexpress neurotrophin-3 (NT-3) and insulin-like growth factor-1 (IGF-1) in SCI rats, which

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alleviated hindlimb spasticity and showed an increasing trend in 5HT2CR expression

. Therefore,in

this study, we evaluated the effects of overexpressing 5HT2CR in motor neurons, inhibitory

interneurons, and excitatory interneurons. A transgenic mouse model with hindlimb spasticity

following SCI, which carries the Cre enzyme gene, was used. By utilizing AAV viruses with DIO

elements, 5HT2CR was selectively overexpressed in different types of neurons. The aim of this

study was to evaluate the differential impact of 5HT2CR overexpression in these three types of

neurons on motor function and spasticity symptoms in SCI mice.

We demonstrated that 5HT2CR overexpression can alleviate spasticity in a SCI mouse model. By

using transgenic mice, we successfully induced 5HT2CR overexpression in various types of spinal

cord neurons. Our findings show that overexpressing 5HT2CR in motor neurons significantly

improves muscle spasticity, motor function, and gait during the chronic phase of SCI, while

overexpressing 5HT2CR in interneurons better alleviates neuropathic pain.

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RESULTS

AAV-Mediated 5HT2CR Gene Overexpression in Target Neurons: Focus on MNs and INs

This experiment is based on the theory of the Cre-LoxP system. Firstly, we engineered an AAV,

designated HBAAV2/BBB-CAG-DIO-m-Htr2c-3xflag-ZsGreen, with the capability to cross the

blood-brain barrier. This AAV vector incorporates both the 5HT2CR gene fragment and a green

fluorescent marker (Figure 1A-B).

To evaluate the effects of 5HT2CR on spasticity, motor function, and sensory processing following

SCI, we divided mice into five groups: the sham surgery group (laminectomy without SCI), the SCI

group, the Chat group (SCI with AAV-mediated overexpression of 5HT2CR in motor neurons), the

VGAT group (SCI with AAV-mediated overexpression of 5HT2CR in GABAergic interneurons),

and the Vglut-2 group (SCI with AAV-mediated overexpression of 5HT2CR in glutamatergic

interneurons). Mice were pre-trained for seven days prior to the induction of the SCI surgery.

100

Following laminectomy at the T9 vertebral level, we generated the SCI model by delivering a

101

vertical impact injury to the T10 spinal cord using a drop-weight device. On the second day post-

102

injury(dpi), the AAV encoding 5HT2CR was diluted to 1.5×10^10 vg/mL (100-fold dilution) using

103

sterile 0.9% saline. This solution was then administered via tail vein injections into ChAT-IRES-

104

Cre, VGAT-IRES-Cre, and Vglut2-IRES-Cre mice (Figure 1A-B).

105

Immunofluorescence was subsequently utilized to co-label ChAT, VGAT, and Vglut-2,

106

demonstrating successful AAV transduction in the target cells. This was evidenced by the

107

examination of transverse sections of the lumbar enlargement spinal tissue, where robust green

108

fluorescent labeling was consistently observed across all experimental groups. At 28 dpi, one mouse

109

from each group was randomly selected for detailed immunofluorescence analysis. Quantitative co-

110

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localization analysis revealed that most of the green fluorescent markers were localized in the

111

expected target cells. These results provide strong evidence of targeted and efficient gene delivery

112

to ChAT, VGAT, and Vglut-2 expressing cells, validating the specificity and effectiveness of the

113

AAV-mediated transduction (Figure 1C).

114

H-reflex tests were conducted at weekly intervals to monitor changes in spasticity in the mice on 35

115

dpi and 56 dpi. Additionally, the locomotor function of the mice was evaluated on both 28 dpi and

116

56 dpi using the Basso Mouse Scale (BMS) to provide quantitative scoring. On 56 dpi, a

117

comprehensive assessment was performed, including Digit Gait Analysis for detailed gait metrics,

118

pain threshold measurements, and the Open Field Test (OFT) to evaluate anxiety and depression-

119

related behaviors. These evaluations collectively provided a thorough assessment of the

120

neurological and functional recovery in the mice (Figure 1D).

121

The Effect of 5HT2CR Overexpression in MNs and INs on Spasticity

122

To evaluate spastic-like behavior, we measured the Hoffman reflex(H-reflex) in the hind limbs of

123

mice. The amplitude of the H-reflex decreases with increasing stimulation frequency under

124

continuous stimulation, a phenomenon known as rate-dependent depression (RDD). We utilized the

125

RDD of the H-reflex to assess the extent of this suppression

. To investigate the changes in spastic-

126

like symptoms in the hind limbs of these SCI mice, RDD of the H-reflex were measured at 35 dpi

127

and 56 dpi.

128

We recorded both the M-wave and the H-wave. The M-wave, which is generated by the activation

129

of motor axons, showed minimal variation among the different groups of mice. While the Ia sensory

130

fibers mediate the excitation of motor neurons upon stimulation, resulting in a delayed H-wave

131

(Figure 1E-G). As the stimulation frequency increases, the amplitude of the H-wave diminishes.

132

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However, in injured mice exhibiting muscle spasms due to excessive excitation of motor neurons,

133

the reduction in H-wave amplitude is suppressed

. Consequently, as the stimulation frequency

134

increases from 0.1 Hz to 5 Hz, animals with mild spasticity show a significant decrease in RDD,

135

whereas animals with severe spasticity exhibit the opposite pattern.

136

In this experiment, the average relative amplitude of the H-wave (H%) in mice decreased when the

137

stimulation frequency reached or exceeded 0.5 Hz. The RDD of H-wave in the SCI group was

138

significantly higher than injured mice in the other three groups which overexpressed 5HT2CR. At

139

35 dpi, the decrease in H-wave amplitude in the SCI group was not significant (RDD decreased

140

from 100% at 0.2 Hz to 89.73 ± 25.99% at 5 Hz). In contrast, the H% in the VGAT, ChAT, and

141

Vglut-2 groups was significantly lower than that in the SCI group (decreasing to 36.70% ± 26.20%,

142

58.59% ± 16.5%, and 46.93% ± 24.90% at 5 Hz, respectively, all P < 0.05 compared to SCI group)

143

(Figure 1G). Similar RDD patterns were observed at 56 dpi, with the SCI group showing 81.8% ±

144

21.80% at 5 Hz, compared to 22.80% ± 21.08% in the VGAT group ,23.80% ± 15.62% in the ChAT

145

group and 50.2%±10.77 % in the Vglut-2 group (Figure 1G).

146

These findings indicate that overexpressing 5HT2CR in both motor neurons and interneurons can

147

significantly reduce the excessive excitation of motor neurons. This, in turn, effectively alleviates

148

spastic symptoms observed in the hind limbs following SCI.

149

5HT2CR Overexpression in Motor Neurons Reduces Anxiety-Depressive Behaviors in SCI Mice

150

After SCI, anxiety and depressive-like behaviors are commonly observed, and these negative

151

emotions may be exacerbated by muscle spasticity and pathological neuropathic pain. Serotonin, an

152

important target for treating anxiety and depression, has rapid antidepressant effects. The OFT is an

153

effective method for measuring locomotor activity and anxiety-like behavior

. Exploration of the

154

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center area is commonly considered an indicator of the anxiety and depression levels in mice. To

155

investigate the impact of overexpression of 5HT2CR in different types of neurons on anxiety

156

behavior following SCI, the OFT was used to observe the effects of 5HT2CR overexpression in

157

motor neurons or interneurons on the emotional state of spinal cord injured mice. The movement

158

trajectories of the mice in the open field were recorded (Figure 2C). The speed and distance traveled

159

by mice through the all area and the center area were evaluated. Meanwhile, attention was paid to

160

the number of times mice passed through the center area and the duration of their traversal in the

161

center area (Figure 2A-B).

162

The traversal velocity and distance across all areas for mice of ChAT, VGAT, and Vglut-2 groups

163

showed no significant improvement compared to those in the SCI group (p > 0.05). Specifically, the

164

Vglut-2 group demonstrated minimal variation across any observed metrics relative to the SCI group,

165

with a notable reduction in velocity through the central area (15.53 cm/s ± 3.82 cm/s, p <

166

0.001). Contrastingly, during traversal of the central area, ChAT group mice exhibited a significantly

167

greater velocity compared to the SCI group(p < 0.05). No significant difference in speed was

168

observed between the ChAT and Sham group (p > 0.05). Mice in the ChAT group traveled 175.31

169

mm ± 17.62 mm, which was significantly greater than the SCI group (83.83 mm ± 6.50 mm ,p

＜

170

0.001) but notably less than the Sham group (911.16 mm ± 164.55 mm ,p

＜

0.05)(Figure 2D-E).

171

The duration time in the center area of the open field for mice in the ChAT group was significantly

172

higher than that for mice in the SCI group (P = 0.0304) (Figure 2F) The ChAT group did not show

173

any significant difference in bouts in the center compared to both the Sham groups(p

＜

0.05) (Figure

174

2G).

175

In summary, the ChAT group mice exhibited better exploratory ability. An effect that was less

176

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pronounced in the VGAT and Vglut-2 groups. These results indicate that overexpression of 5HT2CR

177

in motor neurons alleviates anxiety and depressive-like symptoms in SCI mice. However,

178

overexpression of 5HT2CR in interneurons does not improve anxiety and depressive-like behaviors.

179

Modulation of Motor Function and Gait Dynamics Through 5HT2CR Overexpression

180

SCI results in motor impairments, primarily due to the loss of descending signals from higher central

181

centers following spinal cord transection. Additionally, spasticity further exacerbates these

182

impairments, specifically affecting movement and gait. We performed a multidimensional

183

evaluation of hind limb motor function in mice by integrating gait analysis with the DigiGait system

184

and BMS scoring

27,28

185

Utilizing the DigiGait gait analysis system, we examined the impact of 5HT2CR overexpression in

186

distinct neuronal populations on mouse gait patterns (Figure 2H). Key gait parameters, such as stride

187

time, braking time, stance time, stride frequency, hindlimb stance width, and hindlimb step angle,

188

were rigorously analyzed. Due to poor recovery of motor function after injury, the SCI group mice

189

were unable to complete the gait recording process using the DigiGait system. The results showed

190

that compared to the control group, the Vglut-2 group exhibited significant differences in gait

191

parameters compared to the Sham group, particularly in stance time and braking time. The stance

192

time was significantly prolonged in the Vglut-2 group (P < 0.05 for the right hind limb

，

P < 0.01

193

for the left hind limb). Moreover, braking time was significantly extended in both hind limbs, with

194

a particularly pronounced increase in the left hind limb (P < 0.001), indicating suboptimal regulatory

195

effects in the Vglut-2 group (Figure 2I).

196

In contrast, the ChAT and VGAT groups demonstrated notable improvements across various gait

197

parameters, with no significant abnormalities observed in stance time or stride frequency (Figure

198

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2I). This suggests enhanced motor autonomy and coordination. In addition, the Vglut-2 group

199

showed a significant increase in stance width and hind limb stance angles (P < 0.05), but this did

200

not result in positive gait adjustments. While the ChAT and VGAT groups demonstrated stable

201

performance in these parameters.

202

Overall, the overexpression of 5HT2CR did not effectively improve gait characteristics in the Vglut-

203

2 group, whereas the ChAT and VGAT groups demonstrated superior regulatory effects.

204

At 35 dpi and 56 dpi, motor function was assessed using the BMS scoring system. (Figure 2J). The

205

results revealed no significant difference in BMS scores between the Vglut-2 group and the SCI

206

group (P > 0.05), aligning with the prolonged gait cycle and braking times observed in the gait

207

analysis. This finding further underscores the limited impact of Vglut-2 in modulating gait function.

208

In contrast, mice in the ChAT and VGAT groups exhibited significant improvements in BMS scores,

209

highlighting their superior efficacy in enhancing motor recovery (P < 0.01 and P < 0.05, respectively)

210

(Figure 2K).

211

This outcome is consistent with the marked enhancements observed in gait analysis, indicating that

212

elevating the expression of 5HT2CR in motor neurons or GABAergic interneurons can offer

213

significant potential for recovering gait function and alleviating motor impairments after SCI.

214

Overexpression of 5HT2CR in Glu Interneurons Modulates Mechanical and Thermal Pain

215

Thresholds after SCI

216

Hyperalgesia and abnormal pain can be quantitatively assessed in different mouse groups through

217

thermal and mechanical pain thresholds using the Hargreaves and Von Frey tests, respectively. At

218

55 dpi, thermal pain thresholds were determined across various mouse cohorts. The Vglut-2 group

219

exhibited significantly elevated thermal thresholds relative to the SCI group (P < 0.01). No

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significant differences in thermal pain thresholds were observed between the ChAT and VGAT

221

groups compared to the SCI group (Figure 3A). To reduce potential confounding factors, the Von

222

Frey test was conducted the following day. The results revealed that mechanical pain thresholds in

223

the ChAT, VGAT, and Vglut-2 groups were markedly higher than those in the SCI group (P < 0.0001,

224

P < 0.05, P < 0.001, respectively) (Figure 3B). This data underscores that overexpression of

225

5HT2CR in interneurons more effectively ameliorates pain hypersensitivity in SCI mice.

226

Overexpression of 5HT2CR in MNs or INs regulates the expression levels of GABAAR and KCC2

227

in motor neurons.

228

Immunofluorescence analysis was conducted to assess the expression levels of NMDAR1 and

229

NMDAR2A in the lumbar enlargement of different mouse groups. In the SCI group, the expression

230

levels of both NMDAR1 and NMDAR2A were elevated compared to the Sham group (NMDAR1

，

231

210.25±36.60 for the SCI group, 158.5±23.10 for the Sham group, P > 0.05; NMDAR2A, P < 0.001)

232

(Figure 3C-E). This indicates that SCI induces an abnormal upregulation of NMDAR expression.

233

In the ChAT and VGAT groups, the number of NMDAR1-positive cells did not differ significantly

234

from the Sham group, whereas the Vglut-2 group exhibited a notable increase in NMDAR1

235

expression (p < 0.05). Additionally, the number of NMDAR2A-positive cells in the lumbar

236

enlargement of ChAT and VGAT groups was significantly lower than that in the SCI group (P

＜

237

0.05,P

＜

0.05, respectively, but no significant difference was observed between the Vglut-2 and SCI

238

groups (p > 0.05) (Figure 3C-E). These findings suggest differential regulation of NMDAR subunits

239

in response to SCI and its modulation by various neuronal subtypes.

240

In summary, overexpression of 5HT2CR in motor neurons and GABAergic interneurons of SCI

241

mice significantly reduced the expression of NMDAR1 and NMDAR2A in the lumbar enlargement.

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In contrast, overexpression of 5HT2CR in glutamatergic interneurons had no significant effect on

243

NMDAR expression in SCI mice.

244

The Effects of 5HT2CR Overexpression in different neurons on GABAAR and KCC2.

245

Initially, at 56 dpi, we conducted a Western Blot analysis on the entire spinal cord of mice to measure

246

the expression levels of BDNF, TrkB, KCC2, and GABAAR. The results indicated no significant

247

differences in the expression of BDNF and TrkB between the VGAT, ChAT, Vglut-2, and SCI groups

248

(P > 0.05) (Figure 4A-B). However, the expression levels of GABAAR in the VGAT and ChAT

249

groups were significantly lower compared to the SCI group (p = 0.0025 and p = 0.0001,

250

respectively). Additionally, the expression of KCC2 was significantly reduced in the VGAT and

251

Vglut-2 groups compared to the SCI group (p = 0.021 and p = 0.005, respectively) (Figure 4A-B).

252

Therefore, the overexpression of 5HT2CR does not modulate the BDNF-TrkB pathway.

253

Next, we focused on the lumbar enlargement region of the mouse spinal cord, assessing the

254

expression of GABAAR and KCC2 in motor neurons via immunofluorescence. The mean intensity

255

of these proteins in motor neurons was our primary metric of interest (Figure 4C-D). The results

256

indicated that there were no significant differences in the expression levels of GABAAR and KCC2

257

between the VGAT, ChAT, and Vglut-2 groups when compared to the SCI group (Figure 4E-F).

258

However, both the VGAT and ChAT groups exhibited a trend towards decreased mean intensity of

259

GABAAR and increased mean intensity of KCC2 in motor neurons relative to the SCI group.

260

Conversely, the Vglut-2 group showed an increasing trend in the mean intensity of GABAAR and

261

a decreasing trend in the mean intensity of KCC2 compared to the SCI group (Figure 4E-F). In

262

summary, the overexpression of 5HT2CR regulates the expression and function of GABAAR and

263

KCC2 in the spinal cord.

264

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DISCUSSION

265

Serotonin reuptake inhibitors, a class of antidepressants, have been reported to exacerbate spasticity

266

associated with SCI as early as the last century

29,30

. 5HT2CR has been identified in various neuronal

267

populations. The Cre-LoxP system has been utilized to successfully achieve overexpression of

268

5HT2CR in motor neurons, GABAergic interneurons, and glutamatergic interneurons in SCI mice.

269

These neurons demonstrate the physiological characteristics of spinal motor circuits in vitro. The

270

expression of 5HT2CR, which eventually regulates the function and excitability of motoneurons in

271

the lumbar enlargement of the spinal cord, influences spastic-like symptoms, motor function,

272

neuropathic pain, and even anxiety and depressive moods following SCI. The enhancement of this

273

therapeutic effect suggests that improving the normal excitability of motoneurons is fundamental to

274

alleviating these symptoms.

275

The 5HT2 receptor is a seven-transmembrane G protein-coupled receptor (GPCR) that interacts

276

with G proteins to stimulate phospholipase C (PLC), leading to increased Ca2+ levels

31,32

. 5HT2CR

277

is widely distributed in the CNS and has been the focus of research for the treatment of various

278

diseases. However, intriguingly, the effects of 5HT2CR agonists, antagonists, or inverse agonists

279

have shown inconsistency across multiple studies. For example, SERT, a classic antidepressant, is

280

well-known for its role in mood regulation. Both 5HT2CR agonists and antagonists have been

281

proposed for the treatment of anxiety and depression

33-36

. 5HT2CR inverse agonists and agonists

282

are both considered promising for treating spasticity following SCI. The constitutive activity of

283

5HT2CR and mRNA editing may account for this potential.

284

mRNA editing and constitutive activity of 5HT2CR are two important features that influence its

285

function. mRNA editing of 5HT2CR supports the diversity of 5HT2CR and determines serotonin

286

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signaling and drug therapeutic effects

37,38

. Constitutive activity of 5HT2CR is also related to the

287

extent of RNA editing. RNA editing of 5HT2CR inhibits its constitutive activity

39,40

. The

288

constitutive activity of 5HT2CR allows it to be activated even without ligand binding, meaning its

289

activity is not influenced by agonists. AAV viruses carrying 5HT2C mRNA were injected into SCI

290

mice. We speculate that this treatment may have had an inhibitory effect on the constitutive activity

291

of 5HT2CR.

292

The Effect of 5HT2CR on the Relief of Spasticity and Motor Function

293

5HT2CR can form a complex with GluNR2A, activating Src protein phosphorylation and regulating

294

the depolarization of motor neurons

. Additionally, postsynaptic density protein-95 (PSD-95) has

295

been shown to be involved in the signaling pathway of 5HT2CR. Excitotoxicity mediated by

296

NMDA-PSD95-nNOS severely impacts neuronal function after injury

. Studies have shown that

297

Fluoxetine, by reducing RNA editing of 5HT2CR, can specifically block glutamate receptors and

298

thereby exert neuroprotective effects

43,44

. We confirmed that 5HT2CR is overexpressed in motor

299

neurons and inhibitory interneurons, significantly reducing the expression levels of NR1 and NR2A

300

in the lumbar enlargement of the spinal cord. This result is consistent with the improvement in motor

301

function observed in our behavioral experiments. Therefore, we hypothesize that the overexpression

302

of 5HT2CR in motor neurons or GABAergic interneurons may be related to the improvement in

303

motor function and the alleviation of spasticity, potentially through the downregulation of NMDARs

304

in the anterior horn of the lumbar spinal cord.

305

NMDAR plays a key role in regulating neuronal excitability and synaptic plasticity. After SCI,

306

NMDAR may undergo excessive activation, contributing to pathological processes such as

307

excitotoxicity, abnormal spinal cord circuit remodeling, cellular oxidative stress, and inflammatory

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responses

.The reduction of NMDAR1 and NMDAR2A in the ChAT and VGAT groups likely

309

diminishes excessive excitatory signaling, thereby alleviating spasticity. This is supported by the

310

improved motor function observed in these groups.Although overexpression of 5HT2CR in

311

glutamatergic inhibitory interneurons resulted in some improvement in spasticity in mice, the gait

312

data from the Vglut-2 group did not show significant improvement in walking function. This may

313

be related to the excitatory effects of glutamatergic signaling in spinal reflex regulation. After SCI,

314

the excessive activation of glutamatergic signaling exacerbates the excitability of neural circuits.

315

Motor function, especially coordinated gait, requires a fine balance between excitatory and

316

inhibitory signaling.

317

It is widely recognized that motor neurons, as the final common pathway for voluntary movement,

318

play a direct role in regulating muscle activity

319

The significant benefits observed in the ChAT group suggest that enhancing 5HT2CR signaling in

320

motor neurons may optimize motor output and directly improve gait dynamics. In contrast, the

321

sluggish response in the Vglut-2 group indicates that simply modulating glutamatergic signaling

322

may be insufficient to effectively promote gait recovery. This may stem from the complexity of the

323

glutamatergic network, where excessive activation could disrupt rather than restore motor function,

324

or from compensatory mechanisms that counteract the therapeutic effects of 5HT2CR

325

overexpression.

326

In summary, the ChAT and VGAT groups highlight the important role of 5HT2CR modulation in

327

motor neurons and GABAergic interneurons in influencing motor output and synaptic plasticity.

328

While glutamatergic signaling is crucial for modulating spasticity, its role in gait recovery is more

329

complex compared to the GABAergic and motor neuron-specific mechanisms. This may involve

330

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combinatorial approaches that balance excitation and inhibition while optimizing motor neuron

331

performance. Further studies could provide a more holistic strategy for restoring gait function in

332

spinal cord injury patients.

333

The Expression of 5HT2CR Correlates with the Regulation of Mood and Motor Function

334

The results of the OFT study reveal the differential effects of 5HT2CR overexpression in different

335

neuronal subtypes on anxiety and depression-like behaviors in SCI mice. It was found that the ChAT

336

group mice exhibited stronger exploratory behavior, suggesting alleviation of anxiety and

337

depression-like symptoms. However, in the other two groups of mice, especially the Vglut2 group,

338

the improvement in behavior was not as pronounced. Analysis of gait parameters indicated that

339

5HT2CR overexpression in glutamatergic interneurons did not have an ideal regulatory effect on

340

the mice's motor function. Perhaps the insufficient improvement in motor function led to the lower

341

central area traversal speed observed in the Vglut group during the OFT. This may suggest that the

342

improvement in the mice

’

s anxiety and depressive-like behaviors is influenced by the recovery of

343

motor function. In other words, better motor function helps alleviate the anxiety and depression-like

344

behaviors in mice.

345

Interneurons regulate the activity of neural circuits by releasing inhibitory or excitatory

346

neurotransmitters

.Glutamatergic interneurons are primarily responsible for excitatory signaling

347

within the spinal cord and in ascending spinal regions. Overexpression of 5HT2CR in these neurons

348

may lead to excessive activation of excitatory neural pathways. The imbalance between excitatory

349

and inhibitory signaling in spinal cord neural circuits is closely linked to motor dysfunction,

350

spasticity, and neuropathic pain following SCI

.Although they play an important role in the

351

regulation of neural signaling, their impact on mood regulation is more complex and indirect.

352

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As an SSRI, the activation of 5HT2CR carries the possibility that it not only indirectly modulates

353

mood through its effects on spasticity or pain, but also interacts with neural circuits involved in

354

mood regulation. The serotonergic system is closely linked to these mood-regulating

355

pathways

The activity of motor neurons may also influence the release of neurotransmitters, such

356

as the serotonergic system itself, or other neurotransmitter systems like dopamine and

357

norepinephrine, thereby having a direct impact on mood.

358

In summary, the overexpression of 5HT2CR in motor neurons may improve anxiety and depression-

359

like behaviors in SCI mice through direct and/or indirect mechanisms. However, the direct impact

360

of 5HT2CR overexpression on these behaviors requires further investigation. Additionally, we are

361

aware that AAV vectors may be taken up by brain neurons. A study by Kawabata et al. suggests that

362

AAV2 capsid variants predominantly target cerebral vascular endothelial cells, with a lower

363

transduction efficiency in brain neurons

.We believe that the significant impact on 5HT2CR

364

expression in brain neurons and its potential influence on behavioral outcomes is unlikely.However,

365

we also recognize the importance of acknowledging this limitation in the study. In future research,

366

optogenetic manipulation or fiber-optic recording techniques may be used to more directly observe

367

the relationship between motor neuron activity and mood changes.

368

5HT2CR Overexpression in Glutamatergic Excitatory Neurons Alleviats Neuropathic Pain

369

Mice in the Vglut-2 group were observed to experience milder neuropathic pain, an effect that was

370

not significant in the other two groups. This may be due to the excessive activation of 5HT2CR in

371

glutamatergic excitatory interneurons, which inhibits the presynaptic release of glutamate, reducing

372

the overactivation of pain-related neural circuits. It is reported that inhibite the expression and

373

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function of different NMDAR subtypes can alleviate neuropathic pain after SCI

45,50

.Increasing the

374

expression of KCC2 after SCI has also been considered a potential strategy for alleviating

375

neuropathic pain

.However, these studies did not focus on the diverse roles of different neuronal

376

subtypes in the onset and treatment of pain.

377

Our study may provide support for the idea that targeting and modulating 5HT2CR in glutamatergic

378

excitatory neurons is a promising therapeutic strategy for treating neuropathic pain after SCI. Future

379

research should further explore the underlying mechanisms to identify treatment approaches that

380

enhance analgesic effects.

381

In summary, overexpression of 5HT2CR in glutamatergic excitatory interneurons (Vglut-2 group)

382

can alleviate neuropathic pain following SCI, highlighting the potential of this neuronal subtype for

383

pain treatment.

384

The Therapeutic Effect of 5HT2CR Overexpression is Primarily Mediated by Modulating

385

GABAAR and KCC2, Rather than the BDNF-TrkB Pathway

386

Disruption of chloride homeostasis in motor neurons is thought to be closely linked to the

387

development of spasticity and neuropathic pain after SCI. Research indicates that boosting KCC2

388

activity can help alleviate hyperreflexia and spasticity associated with chronic SCI

. The BDNF-

389

TrkB pathway plays a crucial role in the regulation of KCC2 and the recovery of neuronal

390

function

53,54

. Moreover, the activation of the BDNF-TrkB pathway and the upregulation of KCC2

391

expression play key roles in regulating pain hypersensitivity after SCI, contributing to the alleviation

392

of neuropathic pain

. However, our study revealed that overexpression of 5HT2CR in various types

393

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of neurons does not significantly affect the BDNF-TrkB pathway in the spinal cord, and KCC2

394

expression is suppressed. Nevertheless, this observation is crucial for understanding the scope of

395

the therapeutic effects regulated by 5HT2CR. It may suggest that the overexpression of 5HT2CR in

396

specific neurons can improve motor function and reduce spasticity, but this is not mediated by the

397

BDNF-TrkB pathway, which is typically associated with neural plasticity and neuronal

398

recovery.Surprisingly, results from the ChAT and VGAT groups suggest that overexpression of

399

5HT2CR in motor neurons or GABAergic interneurons might enhance KCC2 function specifically

400

in motor neurons.

401

As previously mentioned, 5HT2CR, as a GPCR, activates PLC to regulate intracellular calcium ion

402

levels. Additionally, changes in KCC2 and GABAAR are crucial for the regulation of intracellular

403

chloride ion concentration. Proper Cl

⁻

levels also play an important role in maintaining the function

404

of KCC2 and GABAAR. The expression and function of postsynaptic GABA receptors also regulate

405

the activity of voltage-gated calcium channels (Cav), thereby influencing the excitability of motor

406

neurons

. After SCI, the hyperpolarizing effect of GABAAR on neurons is reduced, leading to

407

abnormal ion plasticity and promoting the development of spasticity and chronic pain

. This has

408

been shown to be associated with the downregulation of KCC2

. The primary role of KCC2 is to

409

maintain a low level of Cl⁻ concentration within neurons. This function is influenced by GABAAR,

410

and KCC2 can also regulate GABAergic signaling and its receptors. In mature neurons, GABAAR

411

triggers synaptic inhibition, whereas in immature neurons, it induces synaptic excitation. Enhancing

412

the function of KCC2 can facilitate the efflux of intracellular Cl⁻, leading to a hyperpolarizing shift

413

in GABAergic activity, which aids in limiting the occurrence of neuronal damage

58-61

. Serotonergic

414

fibers have a significant impact on the GABAergic system and KCC2

. We found that by increasing

415

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the expression of 5HT2CR after SCI, both VGAT and ChAT groups of mice exhibited upregulation

416

of KCC2 at 56dpi, accompanied by downregulation of GABAAR. This suggests that overexpression

417

of 5HT2CR in motor neurons or GABAergic interneurons can enhance KCC2 function in the lumbar

418

enlargement of the spinal cord while suppressing the expression and function of GABAAR, thereby

419

reversing the excessive excitability of motor neurons following injury. The observed upregulation

420

of KCC2 and downregulation of GABAAR in the ChAT and VGAT groups may enhance inhibitory

421

signaling and reduce neuronal hyperexcitability. These molecular changes are consistent with the

422

reduction in spasticity and improvement in motor function observed in these groups. These findings

423

refine our understanding of the molecular targets of 5HT2CR in the context of SCI recovery.

424

Given the limited efficacy and potential adverse effects of current pharmacological treatments for

425

spasticity and neuropathic pain after SCI, further investigation into targeting 5HT2CR is considered

426

to have important clinical implications. Therapeutic agents targeting 5HT2CR, including agonists,

427

antagonists, and inverse agonists, have been shown to demonstrate some promise in preclinical

428

models. However, these compounds are often characterized by low specificity and are associated

429

with unwanted side effects when administered over extended periods. In contrast, gene therapy has

430

been proposed as a potential strategy for providing more durable and precise treatments. Unlike

431

small-molecule drugs, gene therapy is able to selectively target specific neuronal populations,

432

thereby minimizing off-target effects on other systems. This targeted approach is believed to offer

433

substantial advantages in optimizing therapeutic outcomes and reducing the risks of systemic

434

complications, underscoring the potential of gene therapy as a more effective strategy for SCI

435

management.

436

To sum

，

in this study, we demonstrate that 5HT2CR has significant therapeutic potential for

437

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alleviating spasticity and related complications following SCI. Specifically, overexpression of

438

5HT2CR in motor neurons not only significantly improves motor function but also effectively

439

reduces anxiety and depression symptoms. However, this approach shows limited effectiveness in

440

improving mechanical and thermal pain. In contrast, overexpression of 5HT2CR in interneurons,

441

particularly in Gluergic interneurons, provides better relief from neuropathic pain. Our research

442

offers new theoretical support for 5HT2CR as a drug target for chronic complications following SCI

443

and provides a new perspective to address past controversies regarding the function of 5HT2CR.

444

Given the limited efficacy and potential adverse effects of existing medications for treating

445

spasticity and neuropathic pain after SCI, further research on 5HT2CR holds significant clinical

446

relevance.

447

LIMITATIONS OF THE STUDY

448

Therapeutic effects of 5HT2CR modulation after SCI vary due to differences in constitutive activity,

449

mRNA editing, and neuronal subtype targeting. While AAV-mediated overexpression offers cell-

450

type specificity, the Cre-LoxP strategy enabled selective expression in motor, GABAergic, and

451

glutamatergic neurons, yet variability in viral transduction efficiency and the possibility of off-target

452

or supraspinal uptake cannot be entirely excluded. Improvements in anxiety- and depression-like

453

behaviors may partly reflect secondary benefits from motor recovery, complicating attribution of

454

direct serotonergic modulation. Finally, future studies integrating electrophysiological or

455

optogenetic approaches will be beneficial for establishing causal links between targeted 5HT2R

456

modulation, circuit dynamics, and functional recovery.

457

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RESOURCE AVAILABILITY

458

Lead contact

459

Further information and requests for resources and reagents should be directed to and will be

460

fulfilled by the lead contact, Dr.Liang-jie Du(duliangjie-ccmu@foxmail.com(L.J.D))

461

Materials availability

462

This study did not generate new unique reagents.

463

Data and code availability

464

•

All data reported in this paper will be shared by the lead contact upon request.

465

•

This study does not report original code

466

•

Any additional information required to reanalyze the data reported in this paper is

467

available from the lead contact upon request.

468

ACKNOWLEGMENTS

469

We thank https://Biorender.com for its technical support.We thank the Beijing Municipal Natural

470

Science Foundation (Grant No. 7212140) for funding.

471

AUTHOR CONTRIBUTIONS

472

Conceptualization, H.G. and Z.T. ; methodology, H.G. and Z.T. ; formal analysis, H.G.,Y.Z.S. and

473

Z.M.Y; writing – original draft, H.G.; writing – reviewing and editing, H.G.and Z.T ; visualization,

474

H.G., and R.R.; supervision, X.X.D and L.J.D.; project administration, L.J.D.; funding acquisition,

475

L.J.D..

476

DECLARATION OF INTERESTS

477

The authors declare no competing interests.

478

STAR

★

METHODS .

479

Detailed methods are provided in the online version of this paper and include the following:

480

○ KEY RESOURCES TABLE

481

○ EXPERIMENTAL MODEL AND PARTICIPANT DETAILS

482

•

Mouse strains and genotyping

483

•

Animal surgery and post-surgery care

484

•

Vector Administration

：

Tail vein injections

485

•

Behavioral tests

486

•

In vivo electromyography recordings (H-reflex)

487

•

DigiGait footprint analysis

488

•

Basso mouse scale (BMS)

489

•

Open field test (OFT)

490

•

VonFrey filament test

491

•

Thermal hyperalgesia test

492

•

Western blot analysis

493

•

Tissue processing and Immunofluorescence

494

○ QUANTIFICATION AND STATISTICAL ANALYSIS

495

496

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Compans, B., Camus, C., Kallergi, E., Sposini, S., Martineau, M., Butler, C., Kechkar, A.,

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Klaassen, R.V., Retailleau, N., Sejnowski, T.J., et al. (2021). NMDAR-dependent long-term

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depression is associated with increased short term plasticity through autophagy mediated

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loss of PSD-95. Nat Commun

12, 2849. 10.1038/s41467-021-23133-9.

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Vizi, E.S., Kisfali, M., and L

rincz, T. (2013). Role of nonsynaptic GluN2B-containing NMDA

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receptors in excitotoxicity: evidence that fluoxetine selectively inhibits these receptors and

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10.1016/j.brainresbull.2012.10.005.

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Li, Z., Lu, Y., Liang, S., Li, S., Chen, B., Zhang, M., Xia, M., Guan, D., Verkhratsky, A., and Li,

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B. (2020). Fluoxetine improves behavioural deficits induced by chronic alcohol treatment

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by alleviating RNA editing of 5-HT(2C) receptors. Neurochem Int

134, 104689.

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Gong, H., Xu, X., Talifu, Z., Zhang, C.J., Sun, Y.Z., Yue, Z.M., Rao, J.S., Du, L.J., and Du, X.X.

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(2025). Prospects and challenges in NMDAR signaling in spinal cord injury recovery and

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neural circuit remodeling. Regen Ther

29, 381-389. 10.1016/j.reth.2025.03.008.

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Kanning, K.C., Kaplan, A., and Henderson, C.E. (2010). Motor neuron diversity in

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Anjum, A., Yazid, M.D., Fauzi Daud, M., Idris, J., Ng, A.M.H., Selvi Naicker, A., Ismail, O.H.R.,

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Athi Kumar, R.K., and Lokanathan, Y. (2020). Spinal Cord Injury: Pathophysiology,

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Multimolecular Interactions, and Underlying Recovery Mechanisms. Int J Mol Sci

21.

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Bonthron, C., Burley, S., Broadhead, M.J., Metodieva, V., Grant, S.G.N., Chandran, S., and

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Miles, G.B. (2024). Excitatory to inhibitory synaptic ratios are unchanged at

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presymptomatic stages in multiple models of ALS. PLoS One

19, e0306423.

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Kawabata, H., Konno, A., Matsuzaki, Y., and Hirai, H. (2023). A blood-brain barrier-

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penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted

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AAV2 variant. Mol Ther Methods Clin Dev

29, 81-92. 10.1016/j.omtm.2023.02.016.

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Deng, M., Chen, S.R., and Pan, H.L. (2019). Presynaptic NMDA receptors control

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nociceptive transmission at the spinal cord level in neuropathic pain. Cell Mol Life Sci

76,

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Hasbargen, T., Ahmed, M.M., Miranpuri, G., Li, L., Kahle, K.T., Resnick, D., and Sun, D. (2010).

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cord injury. Ann N Y Acad Sci

1198, 168-172. 10.1111/j.1749-6632.2010.05462.x.

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Bilchak, J.N., Yeakle, K., Caron, G., Malloy, D., and Côté

, M.P. (2021). Enhancing KCC2

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activity decreases hyperreflexia and spasticity after chronic spinal cord injury. Exp Neurol

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Lee-Hotta, S., Uchiyama, Y., and Kametaka, S. (2019). Role of the BDNF-TrkB pathway in

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128, 32-38. 10.1016/j.neuint.2019.04.003.

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Talifu, Z., Pan, Y., Gong, H., Xu, X., Zhang, C., Yang, D., Gao, F., Yu, Y., Du, L., and Li, J.

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(2022). The role of KCC2 and NKCC1 in spinal cord injury: From physiology to pathology.

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13, 1045520. 10.3389/fphys.2022.1045520.

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Tashiro, S., Shinozaki, M., Mukaino, M., Renault-Mihara, F., Toyama, Y., Liu, M., Nakamura,

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M., and Okano, H. (2015). BDNF Induced by Treadmill Training Contributes to the

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Suppression of Spasticity and Allodynia After Spinal Cord Injury via Upregulation of KCC2.

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29, 677-689. 10.1177/1545968314562110.

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Zhao, J., Gao, L., Nurrish, S., and Kaplan, J.M. (2023). Post-synaptic GABA(A) receptors

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potentiate transmission by recruiting CaV2 channels to their inputs. Cell Rep

42, 113161.

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Lu, Y., Zheng, J., Xiong, L., Zimmermann, M., and Yang, J. (2008). Spinal cord injury-

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induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated

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with down-regulation of the chloride transporter KCC2 in rat. J Physiol

586, 5701-5715.

684

10.1113/jphysiol.2008.152348.

685

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Kahle, K.T., Deeb, T.Z., Puskarjov, M., Silayeva, L., Liang, B., Kaila, K., and Moss, S.J. (2013).

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Modulation of neuronal activity by phosphorylation of the K-Cl cotransporter KCC2.

687

Trends Neurosci

36, 726-737. 10.1016/j.tins.2013.08.006.

688

59.

Jarvis, R., Josephine Ng, S.F., Nathanson, A.J., Cardarelli, R.A., Abiraman, K., Wade, F.,

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Evans-Strong, A., Fernandez-Campa, M.P., Deeb, T.Z., Smalley, J.L., et al. (2023). Direct

690

activation of KCC2 arrests benzodiazepine refractory status epilepticus and limits the

691

subsequent neuronal injury in mice. Cell Rep Med

4, 100957. 10.1016/j.xcrm.2023.100957.

692

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Friedel, P., Kahle, K.T., Zhang, J., Hertz, N., Pisella, L.I., Buhler, E., Schaller, F., Duan, J.,

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Khanna, A.R., Bishop, P.N., et al. (2015). WNK1-regulated inhibitory phosphorylation of

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the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons.

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Sci Signal

8, ra65. 10.1126/scisignal.aaa0354.

696

61.

Côme, E., Heubl, M., Schwartz, E.J., Poncer, J.C., and Lé

vi, S. (2019). Reciprocal Regulation

697

of KCC2 Trafficking and Synaptic Activity. Front Cell Neurosci

13, 48.

698

10.3389/fncel.2019.00048.

699

62.

Granger, A.J., Wang, W., Robertson, K., El-Rifai, M., Zanello, A.F., Bistrong, K., Saunders, A.,

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Chow, B.W., Nuñez, V., Turrero Garcí

a, M., et al. (2020). Cortical ChAT(+) neurons co-

701

transmit acetylcholine and GABA in a target- and brain-region-specific manner. Elife

702

10.7554/eLife.57749.

703

63.

Lee, S., Toda, T., Kiyama, H., and Yamashita, T. (2014). Weakened rate-dependent

704

depression of Hoffmann's reflex and increased motoneuron hyperactivity after motor

705

cortical infarction in mice. Cell Death Dis

5, e1007. 10.1038/cddis.2013.544.

706

64.

Toda, T., Ishida, K., Kiyama, H., Yamashita, T., and Lee, S. (2014). Down-regulation of KCC2

707

expression and phosphorylation in motoneurons, and increases the number of in primary

708

afferent projections to motoneurons in mice with post-stroke spasticity. PLoS One

709

e114328. 10.1371/journal.pone.0114328.

710

Journal Pre-proof

STAR

⭐

METHODS

719

KEY RESOURCES TABLES

720

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies
Rabbit monoclonal anti-
BDNF

Abcam

Cat#ab108319,
RRID:AB_10862052

Rabbit monoclonal anti-TrkB Abcam

Cat#ab187041,
RRID:AB_2892613

Rabbit monoclonal anti-
KCC2

Abcam

Cat#ab259969,
RRID

：

N/A

Rabbit monoclonal anti-
ChAT

Abcam

Cat#ab181023,
RRID:AB_2687983

Mouse monoclonal anti-
GABA A Receptor alpha
2/GABRA2

Abcam

Cat#ab193311,
RRID:AB_2890213

Rabbit monoclonal anti-
GAD65+GAD67

Abcam

Cat#Ab183999,
RRID:AB_2890213

Rabbit

recombinant

monoclonal anti-VGLUT2

Abcam

Cat#ab216463,
RRID:AB_2893024

Rabbit

polyclonal

anti-

5HT2CR

Sigma-Aldrich

Cat#SAB4501477,
RRID:AB_10744628

Goat

Anti-Mouse

IgG

Secondary HRP

Bioss

Cat#bs-0296G-HRP,
RRID:AB_10893927

Goat Mouse IgG Secondary
HRP

Bioss

Cat#bs-0296G-HRP,
RRID:AB_10893927

Goat Anti-Rabbit IgG (H+L) ServiceBio

Cat#GB23303,
RRID:AB_2811189

Cy3 conjugated Goat Anti-
mouse IgG

（

H+L

）

ServiceBio

Cat#GB21301,
RRID:AB_2923552

Cy5-labeled goat anti-mouse
IgG

ServiceBio

Cat#GB27301,
RRID:AB_2910251

Alexa

Fluorescent

488-

conjugated Goat Anti-Rabbit
IgG(H+L)

ServiceBio

Cat#GB25303,
RRID:AB_2910224

Bacterial and Virus Strains
pHBAAV-CAG-DIO-MCS-
T2A-ZsGreen

Hanbio Biotechnology

https://www.hanbio.net/produ
ctclass_8/productdetail_11.sht
ml

Experimental Models: Organisms/Strains
C57BL/6 N mice

Beijing Vitong Lihua
Laboratory

https://www.vitalriver.com/#/
animalModel/conventionalAn

Journal Pre-proof

imal?id=46&idd=9&namecod
e=productserve&title

；

129S6-Chat

tm2

（

cre

）

Lowl

mice

Jackson Laboratory

Strain #:006410
RRID:IMSR_JAX:006410

B6J.129S6(FVB)-
Slc32a1

tm2(cre)Lowl

/MwarJ

Jackson Laboratory

Strain #:028862
RRID:IMSR_JAX:028862

B6J.129S6(FVB)-
Slc17a6

tm2(cre)Lowl

/MwarJ

mice

Jackson Laboratory

Strain #:028862
RRID:IMSR_JAX:028862

Software and algorithms
GraphPad Prism 7

N/A

https://www.graphpad.com/sc
ientificsoftware/prism/

SPSS 26.0

N/A

https://www.ibm.com/product
s/spss-statistics

LabChart 8.0

AD Instruments

https://www.adinstruments.co
m/support/LabChart

721

722

EXPERIMENTAL MODEL AND PARTICIPANT DETAILS

723

Mouse strains and genotyping

724

All animal experiments were conducted according to protocols approved by the animal care and use

725

committee at Capital Medical University of Science and Technology(Ethics batch No. AEEI-2021-

726

145). Adult female mice weighing 20-25g were used in this study. After arrival, all mice were

727

acclimated to the animal facility at least 7 days before experiments were initiated. Mice were housed

728

in group as 5 mice per cage and kept in a 12-hour light/12-hour dark cycle at 20-25

℃

room

729

temperature with water and food ad libitum. Only healthy and immunocompetent mice were used.

730

Mice in this study were not involved in any previous procedures.

731

；

129S6-Chat

tm2

（

cre

）

Lowl

/J mice, which express Cre recombinase in cholinergic neurons, without

732

tdisrupting endogenous Chat expression, are referred to as ChAT-IRES-Cre. B6J.129S6(FVB)-

733

Slc32a1

tm2(cre)Lowl

/MwarJ mice, which express Cre recombinase in inhibitory GABAergic neuron

734

cell bodies, without disrupting endogenous vesicular inhibitory aminoacid transporter expression,

735

are referred to as c. B6J.129S6(FVB)-Slc17a6

tm2(cre)Lowl

/MwarJ mice, which express Cre

736

recombinase in glutamatergic neuron cell bodies, without disrupting endogenous vesicular

737

glutamate transporter-2 expression, are referred to as Vglut2-IRES-Cre. All transgenic animals were

738

backcrossed to C57BL/6N for several generations. Heterozygous Cre

mice were used in

739

experiments. Standard genotyping primers are available on the Jackson Lab website. Adult male

740

C57/BL6N mice of SPF grade were obtained from Beijing Vitong Lihua Laboratory Animal

741

Technology Co., Ltd. (Beijing, China).

742

All animals of different strain were randomized using the random number table. The assessments

743

persons were blinded to all the groups and all outcome analysis was carried out by independent

744

investigators blinded to the treatment condition.

745

METHOD DETAILS

746

Animal surgery and post-surgery care

747

The mice were anesthetized with isopentane (3%–4% induction, 1% to 2% maintenance in oxygen).

748

Journal Pre-proof

Until the mice entered deep anesthesia, the limbs of mice were fixed in a prone position on the

749

operating table. Fur was shaved, after skin preparation and disinfection, a longitudinal skin incision

750

was made to separate fascia and muscle before exposing the spinal processes. The spinous process

751

and lamina of T10 verteble was removed, spinal cord was fully exposed. The thoracic 9 and 11

752

vertebra were fixed with a spinal cord impactor (Precision Systems and Instrumentation IH spinal

753

cord impactor, United States) and the exposed spinal cord was impacted with a force of 70

5 kdyn.

754

C57BL/6 N mice were randomly divided into Sham group and SCI group.SCI group accepted the

755

same operation. While the sham group, only laminectomy was performed without hit. After cleaning

756

the operation area, muscle, fascia and skin were sutured layer by layer. At the same time, mice were

757

given 2

U penicillin intraperitoneally to prevent postoperative infection. Mice were placed on

758

a warm blanket throughout operation until wakeup. Five days after surgery, the mice were given

759

penicillin (2

U /day) to prevent infection.

The mice were assisted with urination at least once

760

per 12h until the autonomous urination function was restore

761

Vector Administration

：

Tail vein injections

762

Conditional expression of target genes in Cre- containing neurons was achieved with recombinant

763

adeno-associated viruses (AAVs) encoding a double-LoxP inverted open reading frame (DIO) of

764

target genes. AAV2 is more effective than AAV9 in transducing cell types in the central nervous

765

system

. For chemogenetic experiments with projection targeting, ChAT-IRES-Cre mice(n=6),

766

Vgat-IRES-cre mice(n=6),Vglut2-IRES-cre mice(n=6) were transduced with

HBAAV2/BBB-

767

CAG-DIO-m-Htr2c-3xflag-ZsGreen (15

vg/mL, HanBio,Shanghai, China),divided as ChAT

768

group,Vgat group and Vglut2 group respectively. AAV was diluted with 0.9% saline to a

769

concentration of 1.5

10^10 vg/mL before used. 2 days after the SCI operation, we injected AAV. A

770

total of 20

L of AAV virus solution was injected via tail vein,and the needle was retained 30

771

seconds before withdrawing. For C57/BL6 N mice, we injected 20

L of 0.9% saline 2 dpi after the

772

SCI operation as SCI group(n=6). And the Sham group(n=6),we did not give them

773

injection .Coordinates were slightly adjusted based on mouse age and size. Viral vectors were

774

allowed to express for at least four weeks before behavioral evaluation. Furthermore, seven mice

775

were excluded from the study due to mortality and spinal cord impact deviation.

776

Behavioral tests

777

Behavioral tests were conducted after SCI surgery. At -7 dpi, which means 7 days before SCI surgery,

778

the pre-training assessment will be used to provide the mice with pre-conditioning.We did RDD by

779

electromyography to test spasticity-like behaviors at 35dpi to 56dpi weeks after transplantation.

780

Basso mouse scale (BMS) was measured at 35dpi and 56dpi, DigiGait footprint analysis was

781

measured at 56dpi for gait analysis. Open field test before was performed at 56dpi. Thermal

782

hyperalgesia testand VonFrey test were performed at 55dpi and 56dpi, respectively.

783

In vivo electromyography recordings (H-reflex)

784

The mice were anesthetized with isopentane (3%–4% induction, 1 % to 2% maintenance in oxygen).

785

Two needle electrodes were inserted into the inner side of the lower limb ankle for stimulating the

786

tibial nerve. Two needle-like recording electrodes were inserted into the sole of the foot, a ground

787

electrode was attached to the end of the tail. PowerLab software LabChart (version 8.0, AD

788

Instruments, New South Wales, Australia) was used to convert electrical signals and obtain data. By

789

amplifying the signal (×4000) and filtering it within the range of 300-6k Hz, we stimulate the tibial

790

nerve with a voltage frequency of 0.1Hz and a pulse width of 0.2ms. We measured the maximum

791

H-wave can be achieved with a current of 1mA. Then, to measure the RDD of the H reflex, with a

792

Journal Pre-proof

2-minute interval,20 consecutive stimuli of 0.2, 0.5, 1, 2, and 5 Hz were performed, respectively.

793

The first 5 H-waves of in each group at different stimulus frequencies were discarded, instead of

794

retaining the waves obtained by last 15 stimuli to determine the RDD at different stimulus

795

frequencies

63-65

796

DigiGait footprint analysis

797

Evaluation of hind limb motor function in the SCI mice was assessed by digital footprint analysis

798

system (DigiGaitTM, Mouse Specifics, Inc., Massachusetts, United States). All mice were allowed

799

to walk on the motor-driven belt. The running speed was slowly increased to 5 cm/s. And the foot-

800

prints and body movement were recorded with a high-speed digital video camera from the ventral

801

view of the treadmill belt reflected off the mirror. (Basler high speed camera, Ahrensburg, Germany).

802

Three consecutive gait cycles were evaluated. Three images were recorded for each rat and

803

evaluation.

804

Basso mouse scale (BMS)

805

Mice were trained and adapted to the open field before the experiment. The BMS were assessed

806

35dpi and 56dpi after injury for knowing the locomotor function of mice

. Each examination was

807

conducted by two experienced observers blinded to the group identity. The two researchers

808

performing and recording the test were unaware of the experimental groupings.

809

Open field test (OFT)

810

The exploratory activity and anxiety-like behavior of the mice were evaluated using a Cleversys

811

TopScanLite device (Cleversys, VA, USA)

. During the evaluation, the mcie were placed in the

812

center of the open field (40 × 40 cm), the average velocity, distance traveled and time spent in the

813

center zone by each animal was recorded for 5 min using a high-speed camera. Before the second

814

test, the field area were wiped clean and disinfected with alcohol to prevent the odor of the previous

815

mice from affecting the results.

816

VonFrey filament test

817

The Von-Frey filament test (Aesthesio, RWD, Danmic/USA) was conducted to assess mechanical

818

allodynia in mice at two time points: before and 8 weeks after SCI. Investigators were blinded to

819

the experimental groupings and treatments. Every mouse was placed in a transparent glass box with

820

a raised wire mesh grid and allowed to adjust to the testing environment for 30 minutes. Then,

821

mechanical thresholds were measured by applying a Von-Frey wire (0.04–2.0 g) to the lateral plantar

822

surface of each hind paw. A positive reaction was recorded when the foot retracted, the mice licked,

823

or jumped. The left and right hind limbs underwent six tests, each separated by a 5 minute interval,

824

and the results were then averaged.

825

Thermal hyperalgesia test

826

The Thermal hyperalgesia test was tested to measure thermal injury threshold 1 day after the Von-

827

Frey filament test by a hot plate test (Bioseb, Pinellas Park, FL, United States). The equipment was

828

calibrated to emit heat towards the heat source for determining the baseline withdrawal latency of

829

the animals, which was around 10 seconds. To prevent scalding, the latency limit was established at

830

30 seconds. Positioned beneath the mid-plantar region of the hind paw via a heat conduction plate,

831

a 40W infrared heat source was used. The duration between the start of the stimulus and paw

832

withdrawal was noted for each test. Each mice underwent three evaluations, with each evaluation

833

spaced 10 minutes apart, and the mean value was calculated from the results.

834

Western blot analysis

835

Journal Pre-proof

Mice spine samples were removed under deep anesthesia with sodium pentobarbital. Then freezing

836

it with liquid nitrogen and stored at −80◦C for western blotting. The tissues were used to detect the

837

expression levels of BDNF (ab108319, Abcam, Cambridge, United Kingdom; dilution 1:1000),

838

TrkB(ab187041, Abcam, Cambridge, United Kingdom; dilution 1:5000), GABAA

2 (ab193311,

839

Abcam, Cambridge, United Kingdom; dilution 1:1000) and KCC2(ab25969, Abcam, Cambridge,

840

United Kingdom; dilution 1:1000). Tissues were centrifuged at 12,000 rpm for 10 min at 4◦C after

841

splitting. A BCA protein concentrationassay kit (PC0020, Solarbio, Beijing, China) was used to

842

determine protein concentrations. According to the results,5

loading buffer and PBS were used to

843

adjust the protein concentrations. Then we conducted SDS-PAGE electrophoresis

844

(P0015L,Beyotime,China) and transferred separated proteins to a PVDF (IPVH00010, Millipore,

845

USA) membra. Membranes were then probed with primary antibodies overnight at 4◦C. The

846

following morning, the membranes were washed three times, incubated with a secondary antibody

847

(bs-0295G-HRP, bs-0296G-HRP, Bioss, Woburn, MA, USA dilution 1:3000), washed, and analyzed

848

by chemiluminescence and gel image analysis.All mice were euthanized in accordance with relevant

849

animal ethics guidelines and regulations, ensuring compliance with animal welfare standards.

850

Tissue processing and Immunofluorescence

851

After deeply anesthetized with sodium pentobarbital, the mice quickly entered an anesthetic state

852

and passed away peacefully within a few minutes to minimize pain and distress. The mice were

853

transcardially perfused with 0.9% normal saline.Then, 4% paraformaldehyde solution were used for

854

fixation. Spinal cords from L2 to L6

（

the lumbar enlargement

）

were dissected out, post-fixed at 4

℃

855

overnight then sectioned on a freezing microtome at a thickness of 30 mm. The tissues designated

856

for staining were outlined using a histochemical pen. Subsequently, the sections were treated with

857

3% hydrogen peroxide at room temperature for 25 minutes in the dark, followed by three washes

858

with PBS (pH 7.4). Blocking was carried out using a small amount of 3% BSA before proceeding

859

to incubate the sections with primary antibodies after removing the blocking solution. The primary

860

antibodies including: Chat (ab181923, Abcam, Cambridge, United Kingdom; dilution 1:200),

861

BDNF (ab108319, Abcam, Cambridge, United Kingdom; dilution 1:1000),TrkB(ab187041, Abcam,

862

Cambridge, United Kingdom; dilution 1:5000), GABAA

2 (ab193311, Abcam, Cambridge, United

863

Kingdom; dilution 1:5000) and KCC2(ab25969, Abcam, Cambridge, United Kingdom; dilution

864

1:5000),Vglut2(ab216463, Abcam, Cambridge, United Kingdom; dilution 1:3000),

865

GAD65+67(ab25969,

Abcam,

Cambridge,

United

Kingdom;

dilution

1:5000),

866

NMDA1R(ab109182,

Abcam,

Cambridge,

United

Kingdom;

dilution

1:5000),

867

NMDA2AR(ab174636,

Abcam,

Cambridge,

United

Kingdom;

dilution

1:5000),

868

5HT2CR(SAB45014, Abcam, Cambridge, United Kingdom; dilution 1:1000). After rewarming and

869

incubation with the corresponding. HRP-labeled secondary antibody, the sections were

870

counterstained with DAPI, washed with PBS, and sealed with an autofluorescence quenching agent

871

(G1221, ServiceBio, Wuhan, China). The tissue sections were imaged under a fluorescence

872

microscope (Eclipse C1, Nikon, Tokyo, Japan) and analyzed by strata Quest (Tissue Gnostics,

873

version 7.0.1.176).

874

QUANTIFICATION AND STATISTICAL ANALYSIS

875

The data are presented as mean ± standard error, and statistical analysis was performed using SPSS

876

software (version 26.0, IBM, Armonk, NY, United States). Two independent sample t-tests were

877

employed for pairwise group comparisons, while one-way ANOVA was utilized for comparisons

878

among multiple groups, with Bonferroni’s post hoc test for correction.For data that do not follow a

879

Journal Pre-proof

normal distribution, non-parametric tests were employed: Mann-Whitney U test (for two-group

880

comparisons) and Kruskal-Wallis test (for multiple group comparisons). Statistical significance was

881

defined as p < 0.05.

882

883

884

Figure 1. Selective Overexpression of 5HT2CR in Spinal Neuronal Populations and Its Impact

885

on H-Reflex Modulation

886

(A)Experimental Groups

887

(B)Illustration of viral constructure and the genetic cross strategy using ChAT-IRES-Cre,

888

VGAT- IRES-Cre, and Vglut2- IRES-Cre mice.

889

(C)Representative confocal images showed motoneurons

、

GABAergic interneurons or

890

glutamatergic interneurons

transfected with AAV in

lumbar enlargement

of spinal cord.

891

(D) Diagram of Experimental Design

892

(E) Principle of the H-reflex.

893

(F)Classic electrophysiological trace.

894

(G)RDD of the H -reflex at various stimulus frequencies from 0.2Hz to 5Hz, comparing SCI,

895

VGAT-Cre, CHAT-Cre, and Vglut-2-Cre groups at different time points (35dpi,56dpi).n=5 in

896

each group.

897

*p < 0.05, **p < 0.01

，

***p<0.001 for VGAT vs. SCI,

p<0.05,

p<0.01,

$$$

p<0.001 for ChAT vs.

898

SCI;

p<0.05,

p<0.01,

###

p<0.001 for VGluT2 vs. SCI. Data are presented as means

SEM.

899

900

901

Figure2 Behavioral analysis of mice in different experimental groups.

902

(A-B) Schematic of the open field test setup used to evaluate anxiety-like behavior.

903

904

experimental group.

905

(D-G)

Distance traveled, velocity, total movement, and central zone time were assessed in

906

different experimental groups. n=3 in each group. *p < 0.05, **p < 0.01

，

***p<0.001. ns

907

indicates no statistical difference between groups.

908

(H) Schematic of the Digi Gait system analysis setup used to evaluate locomotor activity.

909

(I)Top:

Right hindlimb stride time, braking time, stance time, and stride frequency, hindlimb

910

stride frequency; hindlimb stance width. Bottom: Left hindlimb stride time, braking time,

911

stance time, and stride frequency, hindlimb stride frequency; hindlimb stance angle. n=5 in

912

each group. *p < 0.05, **p < 0.01

，

***p<0.001.ns indicates no statistical difference between

913

groups.

914

(J) Schematic of the BMS used to evaluate locomotor activity.

915

(K)Top: BMS at 35dpi.Bottom: BMS at 56dpi.n=5 in each group. *p < 0.05, **p < 0.01

，

916

***p<0.001 for ChAT

，

VGAT

，

Vglut-2 group vs SCI group

；

p<0.05,

p<0.01,

###

p<0.001 for

917

ChAT

，

VGAT

，

Vglut-2 group vs Sham group. ns indicates no statistical difference between

918

groups.

919

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Data are presented as means

SEM.

920

Figure3 Pain Assessment and Expression of NMDAR1 and NMDAR2A in Spinal Cord Tissue

921

(A)

Left: Representative schematic of the Hargreaves test used to measure the thermal pain

922

threshold. Right: Latency to thermal pain in different groups. n = 6 in each group.*p < 0.05,

923

**p < 0.01

，

***p<0.001 for SCI,ChAT,VGAT,Vglut-2 group vs. SCI

，

p<0.05,

p<0.01,

924

###

p<0.001 for ChAT,VGAT,Vglut-2 group vs. Sham. n=6 in each group.Data are presented as

925

means

SEM.

926

(B)

Left: Representative schematic of the Von Frey test used to measure the mechanical pain

927

threshold. Right: Paw withdrawal threshold to mechanical pain in different groups. n = 6 in

928

each group.*p < 0.05, **p < 0.01

，

***p<0.001 for SCI,ChAT,VGAT,Vglut-2 group vs. SCI

，

929

p<0.05,

p<0.01,

###

p<0.001 for ChAT,VGAT,Vglut-2 group vs. Sham. n=6 in each

930

group.Data are presented as means

SEM.

931

(C-D)Immunofluorescence staining for NMDAR1 and NMDAR2A punctae on lumbar motor

932

neurons in the SCI group, ChAT group, VGAT group, and VGLUT-2 group.n=3 mice in each

933

group at 8 weeks.The inserts were magnified to show the anterior horn of spinal cord .Scale

934

bar: 50 mm.

935

(E)Quantification of NMDAR2 and NMDAR2A positive punctae on lumbar motor neurons

936

shown in (C-D) . n=3 mice in each group at 8 weeks.Data are presented as means

SEM.

937

*p < 0.05, **p < 0.01

，

***p<0.001.Data are presented as means

SEM.

938

939

Figure 4: Expression and Localization of Neurotransmitter Receptors and Transporters in

940

Spinal Cord Tissue

941

(A-B) Immunofluorescence staining for KCC2 and GABAAR punctae,as well as co-labeling of

942

ChAT,showing the immunofluorescence intensity on lumbar motor neurons in the SCI group,

943

ChAT group, VGAT group, and VGLUT-2 group.n=4 mice in each group at 8 weeks.The inserts

944

were magnified to show the anterior horn of spinal cord .Scale bar: 50 mm.

945

(C-D) Quantification of immunofluorescence intensity for GABAAR and KCC2 on MN shown

946

in (A) and (B). n=3 mice in each group at 56pdi.Data are presented as means

SEM.

947

ns indicates no statistical difference between groups.

948

(E) Representative western blot of BDNF, TrkB, KCC2 ,and GABAAR of spinal cord

949

tissue,showing the expression levels of those protein in the SCI group, Sham group,ChAT

950

group, VGAT group, and VGLUT-2 group.GAPDH was used as a loading control. n=3 mice in

951

each group at 8 weeks.

952

(F)Quantification of those protein levels between the four groups,normalized to GAPDH. n=3

953

mice in each group at 8 weeksData are presented as means

SEM.*p < 0.05, **p < 0.01

，

954

***p<0.001.Data are presented as means

SEM.

955

956

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ChAT

Cre

ChAT-IRES-Cre mouse
VGAT-IRES-Cre mouse
VGLUT2--IRES-Cre mouse

AAV with gene of 5HT2CR by loxP

VGAT

Cre

VGALUT-2

Cre

m-HT2C

ZsGreen

loxp

lox2272

5HT2CR overexpression in MNs or GABA INs or Glu INs

Viral DNA

Capsid

Spinal cord injury at T10

SCI

HBAAV2/BBB-CAG-DIO-m-

Htr2c-3xflag-ZsGreen

SCI+Overexpression 5HT2CR

Sham

M-Wave

H-Wave

Spinal Cord

Muscle

α-motor axon

Electic stimulus

Ia sensory fiber

Pre-train

0dpi

AAV tail vein injection

Spinal Cord Injury

-7dpi

2dpi

35dpi

BMS

H-Reflex Test

56dpi

BMS

Digit Gait Analysis

Von Frey Test

Open field Test

H-Reflex Test

Heat Pain Test

55dpi

Journal Pre-proof