Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury

Severe spinal cord injury in adults leads to irreversible paralysis below the lesion. However, adult rodents that received a complete thoracic lesion just after birth demonstrate proficient hindlimb locomotion without input from the brain. How the spinal cord achieves such striking plasticity remains unknown. In this study, we found that adult spinal cord injury prompts neurotransmitter switching of spatially defined excitatory interneurons to an inhibitory phenotype, promoting inhibition at synapses contacting motor neurons. In contrast, neonatal spinal cord injury maintains the excitatory phenotype of glutamatergic interneurons and causes synaptic sprouting to facilitate excitation. Furthermore, genetic manipulation to mimic the inhibitory phenotype observed in excitatory interneurons after adult spinal cord injury abrogates autonomous locomotor functionality in neonatally injured mice. In comparison, attenuating this inhibitory phenotype improves locomotor capacity after adult injury. Together, these data demonstrate that neurotransmitter phenotype of defined excitatory interneurons steers locomotor recovery after spinal cord injury. Bertels et al. identify that spinal neurons switch neurotransmitter phenotype from excitation to inhibition after spinal cord injury. Manipulation of neurotransmitter phenotype reveals that maintaining excitatory phenotype is essential for locomotor recovery.

S evere spinal cord injury disrupts connections between the brain and the spinal cord, which leads to the inability of body movements controlled by the spinal cord below the lesion. Consequently, spinal circuits require pharmacological agents, electrical stimulation or perineal/tail pinching to generate movements in adult rodents [1][2][3] and electrical stimulation in humans 4-6 after a functionally complete spinal cord lesion. However, these are only temporary aids to enable movement generation and do not overcome the obstacle of regaining long-term mobility after injury.
Intriguingly, although complete spinal cord lesion leads to paralysis in adult rodents, the same injury given to neonatal rodents results in a strikingly proficient hindlimb locomotor ability in adulthood, without any descending axon regeneration across the lesion or any form of external stimulation [7][8][9] . This ability to 'walk without the brain' involves spinal-cord-autonomous functions associated with increased excitability of spinal circuits below the lesion 10,11 , but the underlying mechanisms remain elusive. In addition, how the same insults imposed at different ages lead to divergent locomotor recovery is unknown, even though insights on these processes likely play a pivotal role in developing interventions that enhance locomotor recovery after spinal cord injury in the adult.
Studies with pharmacological perturbation suggest that regulation of excitation-inhibition (EI) balance of circuits below the lesion is a critical mechanism that defines the motor capacity of spinal-cord-injured cats and rodents, regardless of the age of injury [12][13][14] . Along with an injury-induced change in intrinsic motor neuron (MN) properties 15,16 , the balance of synaptic input to MNs is one of the determinants of MN excitability. Although severe injury increases inhibitory synaptic input to MNs innervating limb muscles below the lesion, long-term locomotor training reverses the increased inhibitory drive on MNs derived from the local interneurons [17][18][19] . This activity-dependent reorganization of EI input ratio from spinal interneurons to MNs likely contributes to locomotor improvement after long-term training. Furthermore, neuronal activity can trigger long-lasting modification of gene expression profiles that alter neurocircuit functions in a cell-type-specific manner, including neurotransmitter (NT) phenotype switches [20][21][22][23] . However, little has been explored on whether such activity-dependent mechanisms steer functionality of the spinal cord after injury and how age of injury shapes circuit reorganization.
After a complete spinal cord injury, somatosensory feedback is the only external input to the spinal circuits below the lesion. Among somatosensory populations, proprioceptive afferents (PAs) directly contact MNs and multiple classes of spinal interneurons to transmit movement-matched information necessary for motor control [24][25][26][27] . PA activity is essential for spinal circuit assembly and function during development 28,29 and locomotor recovery after an incomplete spinal cord injury 30 . Given the role of proprioception in circuit development, motor control and recovery, PAs are in a prime position to direct locomotor circuits to a functional network after spinal cord injury in the absence of the brain.
In this study, we found that subsets of excitatory, but not inhibitory, spinal interneurons mediate the age of injury-dependent synaptic connectivity profiles to MNs. NT phenotype specification regulates spatially defined subsets of excitatory interneurons to maintain glutamatergic identity after neonatal injury and to gain inhibitory phenotype after adult injury. Furthermore, disrupting their NT identity from excitatory to inhibitory after neonatal injury abolishes the ability to walk without the brain, demonstrating that preserving the glutamatergic phenotype is essential. Using an intersectional genetic model, we found that removal of PAs disrupts glutamatergic phenotype of the subsets of excitatory interneurons after neonatal injury, indicating that continued glutamatergic phenotype expression is dependent on PA activity. Lastly, genetically attenuating inhibitory phenotype after an adult injury significantly improved defined locomotor parameters, especially when combined with locomotor training. Together, our study reveals age of injury-dependent flexible NT phenotype expression of specific

Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury
Hannah Bertels 1,2 , Guillem Vicente-Ortiz 1,2 , Khadija El Kanbi 1,2 and Aya Takeoka 1,2,3 ✉ Severe spinal cord injury in adults leads to irreversible paralysis below the lesion. However, adult rodents that received a complete thoracic lesion just after birth demonstrate proficient hindlimb locomotion without input from the brain. How the spinal cord achieves such striking plasticity remains unknown. In this study, we found that adult spinal cord injury prompts neurotransmitter switching of spatially defined excitatory interneurons to an inhibitory phenotype, promoting inhibition at synapses contacting motor neurons. In contrast, neonatal spinal cord injury maintains the excitatory phenotype of glutamatergic interneurons and causes synaptic sprouting to facilitate excitation. Furthermore, genetic manipulation to mimic the inhibitory phenotype observed in excitatory interneurons after adult spinal cord injury abrogates autonomous locomotor functionality in neonatally injured mice. In comparison, attenuating this inhibitory phenotype improves locomotor capacity after adult injury. Together, these data demonstrate that neurotransmitter phenotype of defined excitatory interneurons steers locomotor recovery after spinal cord injury.

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classes of excitatory interneurons as critical neuronal substrates that steer locomotor capacity after severe spinal cord injury.

results
Adult mice with neonatal injury exhibit proficient hindlimb locomotion. To determine the age of injury-dependent locomotor capacity, we subjected wild-type mice to a complete spinal cord transection (cSTX) at low thoracic level (~T10) either at postnatal day 5 (P5) or as an adult (~P60). We used ten high-speed cameras to quantitatively characterize hindlimb kinematics of each group together with intact controls during bipedal treadmill locomotion, all at 3-4 months of age ( Fig. 1a-d). Although intact mice generated stereotypical alternating locomotion, mice that received cSTX as an adult (adult cSTX) exhibited complete and permanent paralysis of hindlimbs, examined up to 5 months after injury (Fig. 1b,c). In contrast, adult mice that received cSTX at P5 (P5 cSTX) displayed proficient hindlimb stepping without any externally applied stimulation ( Fig. 1d and Supplementary Video 1).
We assessed hindlimb kinematics by high-resolution quantitative locomotor analyses 30 . We computed 102 parameters (Supplementary Table 1) and applied principal component (PC) analysis to all measured parameters/gait cycle/mouse to identify variability related to differences among intact, adult cSTX and P5 cSTX groups. The largest source of variance (PC1; 37%) was expectedly the difference between hindlimb dragging and stepping ( Fig. 1e; that is, adult cSTX mice contrasting intact and P5 cSTX groups), with parameters related to persistent hindlimb dragging showing high correlation to PC1 (Extended Data Fig. 1a). In addition, the PC distance between intact or P5 cSTX groups to the adult cSTX group did not differ significantly (Fig. 1f). Therefore, we conclude that mice with neonatal injury walk without the brain with proficiency mostly indistinguishable from intact mice.
Locomotor proficiency detected in P5 cSTX mice is not due to establishing connections from the brain via axon regeneration across the complete lesion. We retrogradely labeled descending neurons projecting to the lumbar spinal cord in intact and adult mice with neonatal injury (Extended Data Fig. 1b). In intact mice, injection of G-deleted rabies to the lumbar segments labeled descending projection neurons originating from the brain and spinal cord (Extended Data Fig. 1c). In contrast, we did not detect rabies ON neurons either in the spinal cord above the lesion or in the brain of P5 cSTX mice in adulthood, despite no obvious difference in neuronal infection efficiency within the local circuits between the two groups (Extended Data Fig. 1d). Furthermore, complete re-transection of the spinal cord a few segments rostral to the original lesion did not deteriorate locomotor patterns (Extended Data Fig. 1e,f and Supplementary Video 2). PC analysis applied to kinematic parameters recorded at before and after re-transection revealed that the largest source of variability was an inter-subject variability and not associated with the re-transection (Extended Data Fig. 1f). Therefore, the locomotor ability of adult mice with neonatal injury is attributed to the functionality of spinal circuits completely isolated from the brain.
vGlut2 ON neurons undergo NT phenotype switch after adult injury. Synaptic input from spinal interneurons to MNs is one of the factors regulating the final central nervous system (CNS) output that controls muscle contractions. As inhibitory synaptic input to MNs is known to increase after injury, we asked whether the

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EI synaptic input ratio from spinal interneurons to MNs that innervate hindlimb muscles depends on injury age. To visualize excitatory and inhibitory synaptic terminals, we crossed major excitatory or inhibitory NT Cre-driver lines (vGlut2 cre or vGAT cre ) with Tau LSL-nlsLacZ-SynGFP reporter mice to conditionally express synaptophysin tagged with green fluorescent protein (GFP) (SynGFP ON ) in either excitatory or inhibitory spinal interneurons. This approach indelibly labeled synaptic terminals derived from either genetically glutamatergic vGlut2 ON or inhibitory vGAT ON interneuron populations (Fig. 2a). This method shows high fidelity between genetically labeled excitatory or inhibitory terminals to assess protein expression of vGlut2 or vGAT with immuno-labeling (designated as vGlut2 + or vGAT + , as opposed to genetically labeled vGlut2 ON or vGAT ON ) at the junction of choline acetyltransferase ON (ChAT ON ) MNs in intact spinal cords (>80%; Fig. 2b-d).
Surprisingly, we detected no increase in the number of synaptic terminals derived from genetically marked inhibitory terminals in vGAT cre ::Tau LSL-SynGFP spinal cords after adult injury (Fig. 2c). Instead, we detected a five-fold increase of genetically marked excitatory terminals with vGAT + expression (% SynGFP ON /vGAT + ) in vGlut2 cre ::Tau LSL-SynGFP spinal cords where almost half of the synaptic terminals derived from vGlut2 ON neurons became vGAT + (Fig. 2d,e). This change accompanied the expression of GABA in terminals and post-synaptic inhibitory markers by MN, adjacent to apposition   apposing MNs (left) and % antibody labeling of NT expression (vGlut2 + or vGaT + ) with SynGFP ON genetic markings to visualize NT phenotype (right) of intact and adult cSTX spinal cords. Each dot represents one MN (>10 reconstructed MNs per NT analysis with antibody, per mouse, per genotype, n = 5 for each group, P > 0.05 for both vGlut2 cre and vGAT cre ::Tau LSL-SynGFP intercrosses, two-sided unpaired t-test). SynGFP ON density is normalized to the surface area of measured ChaT ON MNs. a low level of NT phenotype switch is detected in intact preparation for both intercrosses. Mean density of SynGFP ON /vGaT + from each MN in vGlut2 cre ::Tau LSL-SynGFP mice: intact 3.0 × 10 -3 ± 0.7 terminals per μm 2 ; adult cSTX 15.8 × 10 -3 ± 0.4 terminals per μm 2 (P < 0.001, two-sided unpaired t-test, and P < 0.001 for percentages of intact versus adult cSTX in vGlut2 cre ::Tau LSL-SynGFP ). e, Genetically marked SynGFP ON terminals co-immunolabeled for vGaT protein (vGaT + ) in vGlut2 cre ::Tau LSL-SynGFP spinal cord after adult injury. Error bars, s.e.m. Scale bar, 3 µm. D, dorsal; mo, month; NS, not significant; V, ventral.

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sites (Extended Data Fig. 2a,b). In parallel, we found a decrease in % of SynGFP ON /vGlut2 + while the total number of genetically marked SynGFP ON terminals remained unchanged (Fig. 2d). Supporting this observation, we also found that vGlut2 + or vGAT + expression at SynGFP ON terminals is mutually exclusive (Extended Data Fig. 2c,d).
A phenomenon of activity-dependent NT phenotype switch in the brain was reported previously 22,31 . To determine whether this observation at synaptic terminals is the case and, if so, whether the NT phenotype switch occurs uniformly among glutamatergic interneurons, we used in situ hybridization of vGlut2 (Slc17a6) and vGAT (Slc32a1) mRNA. In an intact spinal cord, we detected neurons with a singular expression of vGlut2 or vGAT transcripts in equal abundance with only a minor proportion of neurons co-expressing vGlut2 and vGAT transcripts, distributed sparsely along the dorsoventral axis (Fig. 3a-c and Extended Data Fig. 2e). Together with the protein level analysis where we detected a low level of the opposite NT expression in genetically marked terminals ( Fig. 2c,d), this result demonstrates that interneurons exhibit flexible NT phenotype in an intact spinal cord. Adult cSTX significantly increased the density of neurons with vGlut2/vGAT co-expression in the dorsal and intermediate lamina (Fig. 3b,c). This shift paralleled a decrease in neurons with a singular expression of vGlut2 but no change in vGAT (Fig. 3c), a finding that suggests that a fraction of dorsal and intermediate excitatory interneurons acquires vGAT phenotype at the transcript level after adult injury.
Although co-expression of two distinct NT types by spinal MNs has been reported 32,33 , NT phenotype switch is a previously unknown mechanism in the mammalian spinal cord. Notably, NT phenotype switch not only refers to a complete replacement of one NT by another but can also refer to dynamic changes in the level of two or more NTs at the transcript and protein levels within a single neuron 23 . In addition, neurons can synthesize two or more NTs and release each NT selectively from different pre-synaptic terminals 34,35 . This is consistent with our observation, in which vGlut2 and vGAT transcripts are co-expressed at the cell body, whereas protein

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accumulation at synaptic terminals is exclusive to either vGlut2 or vGAT (Fig. 3d). These findings demonstrate that the previously described increase in inhibitory input to MNs after adult injury 17,18 is not due to sprouting of synapses derived from inhibitory interneurons ( Fig. 2c) but, rather, to a NT phenotype switch of excitatory interneurons (Fig. 2d). Together, our results identified cell-type-specific NT phenotype switch as a pathophysiological response to a severe spinal cord injury affecting the mature nervous system.
vGlut2 ON neurons undergo synaptic sprouting after neonatal injury. Next, we determined whether a similar NT phenotype switch occurred after neonatal injury. Much in contrast, we found no evidence of NT phenotype switch after neonatal injury. Instead, we observed a significant increase in genetically marked glutamatergic SynGFP ON terminals to MNs in vGlut2 cre ::Tau LSL-SynGFP mice ( Fig. 4a,b). This increase is due to synaptic sprouting in response to injury but not injury-induced disruption of synaptic pruning that often occurs during circuit development 36 , because we found a significantly lower number of SynGFP ON inputs to MNs in intact spinal cords at P5 than in spinal cords upon neonatal injury (Fig. 4b).
Together, the two injury models revealed that the age of injury defines two opposing synaptic connectivity profiles of excitatory interneurons to MNs. We found NT switch after adult injury, which led to high vGAT + synaptic input to MNs in contrast to genetically marked glutamatergic synaptic sprouting after neonatal injury (Fig. 4c). These observations raise questions about cell type specificity among excitatory interneurons that participate in synaptic connectivity reorganizations, which circuit components direct these plasticity mechanisms and whether NT phenotype specification determines locomotor capacity after injury. vGlut2 ON synaptic reorganization to MNs is subpopulation specific. vGlut2 and vGAT transcript expression after adult injury reveals that specific vGlut2 ON neurons residing in the dorsal and intermediate lamina are driving the process of NT phenotype switch (Fig. 3b). To gain genetic access to different subpopulations of excitatory interneurons residing along distinct dorsoventral positions in the spinal cord (Fig. 5a), we used stratification by developmental origin 37 . We used three transgenic mouse lines, each expressing Cre-recombinase under the control of a different progenitor domain (PD)-specific transcription factor (PD cre ; Fig. 5b) and intercrossed with the Tau LSL-SynGFP reporter line to selectively visualize synaptic output derived from the selected PD population.
We found that both PD identity and age of injury determined the NT profile of their synaptic output. After the adult injury, Tlx3 ON (dI3 and dI5) and Shox2 ON (V2a), but not Sim1 ON (V3), interneurons exhibited increased SynGFP ON /vGAT + and decreased SynGFP ON / vGlut2 + terminals to MNs (Fig. 5c). In parallel, we performed gene expression analysis using multiplex in situ hybridization of vGlut2 and vGAT mRNA specifically on genetically labeled Tlx3 or Shox2 neurons (Extended Data Fig. 3a,b). In intact spinal cords, we detected that most Tlx3 ON or Shox2 ON neurons displayed singular expression of vGlut2 transcripts, with a minor proportion of neurons with a singular expression of vGAT transcripts or co-expression of vGlut2 and vGAT transcripts. In contrast, we found a significantly higher    Fig. 2c,d. a, P > 0.05, two-sided unpaired t-test. b, Mean of density computed from each MN: SynGFP ON /vGlut2 + : intact 3.2 × 10 -2 ± 0.5 terminals per μm 2 , P5 cSTX 4.6 × 10 -2 ± 0.2 terminals per μm 2 , P5 intact 3.3 × 10 -2 ± 0.2 terminals per μm 2 (P < 0.001, two-sided unpaired t-test); SynGFP ON /vGaT + : intact 3.0 × 10 -3 ± 0.2 terminals per μm 2 , P5 cSTX 4.5 × 10 -3 ± 0.4 terminals per μm 2 , P5 intact 2.4 × 10 -3 ± 0.1 terminals per μm 2 (P > 0.05, one-way aNOVa with Tukey's post hoc test). c, Summary diagram of NT phenotype switch and synaptic sprouting of genetically labeled vGlut2 ON interneurons. Error bars, s.e.m.  After neonatal injury, all three PD populations maintained their glutamatergic phenotype at protein and transcript levels, consistent with the analysis on pan-vGlut2 ON interneurons ( Fig. 4b and Extended Data Fig. 3a,b). Interestingly, we found that the neonatal injury-dependent synaptic sprouting was also subpopulation specific (Fig. 5c). We detected more SynGFP ON boutons from Tlx3 ON and Shox2 ON , but not Sim1 ON , interneurons to MNs in P5 cSTX compared to the intact spinal cord. Together, the age of

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injury-specific NT identity switches and connectivity rearrangements to MNs occur with remarkable specificity among excitatory interneurons.

PA ablation disrupts vGlut2 identity after neonatal injury.
Excitatory interneurons that undergo plastic synaptic sprouting and NT phenotype switches reside primarily within the dorsal and intermediate, but not in the ventral, spinal cord. Under normal circumstances, these neurons receive high vesicular glutamate transporter 1 (vGlut1) ON somatosensory input (Fig. 6a). Given the role of PAs in circuit development and motor recovery, we probed whether PAs might maintain vGlut2 phenotype after neonatal injury. We used an intersectional approach, an intercross of Parvalbumin (PV) cre and Advillin iDTR mouse lines, to express human diphtheria toxin (DTX) receptor (DTR) in PV ON /Advillin ON somatosensory afferents 30 . This approach enabled ablation of PAs with DTX at the time of injury at P5 (P5 cSTX+PA DTX ), which led to a substantial reduction of central PAs marked by vGlut1 ON terminals while leaving the minor non-proprioceptive somatosensory vGlut1 ON terminals unaffected ( Fig. 6b and Extended Data Fig. 4a-c). In contrast to P5 cSTX mice, P5 cSTX+PA DTX mice expectedly showed severe locomotor impairments, without any obvious unspecific neuronal degeneration within local circuits (Extended Data Fig. 4d-f).
To determine whether PA ablation leads to NT phenotype switch after neonatal injury, we reconstructed the spatial distribution of vGlut2 and vGAT mRNA in P5 cSTX, P5 cSTX+PA DTX and intact+PA ablation alone at P5 (intact+PA DTX ) groups. Although P5 cSTX alone does not alter the transcript ratio, P5 cSTX combined with PA ablation showed a marked increase in neurons with vGAT/vGlut2 co-expression in the dorsal and intermediate lamina, like the adult cSTX group (Fig. 6c-e). This shift paralleled a decrease in neurons with a singular expression of vGlut2 but no change in a singular expression of vGAT (Fig. 6e). Because the EI transcript profile in intact+PA DTX resembled that of intact and P5 cSTX alone groups (Fig. 6d,e), we conclude that combining the lesion and PA ablation shift EI gene expression profiles after neonatal injury.
Together, we found that PAs are necessary to sustain a singular expression of vGlut2 by a subset of vGlut2 neurons located mainly in the dorsal and intermediate lamina after neonatal injury. In contrast, the NT phenotype of ventral vGlut2 neurons that receive less PA input appears to be independent of PA activity. The similarity in the spatial distribution of vGAT/vGlut2 double-positive neurons detected in P5 cSTX+PA DTX to those of adult cSTX spinal cords suggests that these double-positive neurons are most likely the same pool of interneurons that undergo a NT phenotype switch after adult injury.
Overexpression of vGAT disrupts proficient locomotion after neonatal injury. PA activity-dependent NT phenotype of spinal excitatory interneurons after neonatal injury raised a question of whether maintaining vGlut2 ON phenotype is essential for proficient stepping without the brain. To establish this link, we performed an intraspinal injection of LoxP-flanked AAVs in the lumbar spinal cord to express vGAT (AAV-DIO-vGAT-Tag) by excitatory neurons (Fig. 7a). As our vGlut2 subpopulation-specific analysis (Fig. 5) does not exhaustively examine other dorsal and intermediate vGlut2 subpopulations (that is, dI1, dI2 and V0 G PDs; Fig. 5b), we chose to use vGlut2 cre ::Tau LSL-nlsLacZ-SynGFP mice for this experiment while establishing inclusion criterion with 90%> infected vGlut2 ON neurons are restricted within dorsal and intermediate lamina. This approach allows us to simultaneously manipulate both Tlx3 cre and Shox2 cre populations as well as other dorsal and intermediate vGlu-t2 ON interneurons that may also contribute to recovery after neonatal injury (Fig. 5b). Broad bilateral injection of the virus across lumbar segments led to expression of the Tag at the cell body and mutually exclusive expression of vGAT + and vGlut2 + at SynGFP ON terminals (that is, a proportional increase in vGAT + expression paralleled by a decrease in vGlut2 + expression; Extended Data Fig.  5a-e). We then examined the locomotor ability of P5 cSTX mice with AAV-DIO-vGAT-Tag (P5 cSTX+vGAT) or control injection of AAV-DIO-Tag (P5 cSTX+control) at ~P60 (Fig. 7a).
Virus-mediated expression of vGAT by dorsal and intermediate excitatory interneurons after neonatal injury deteriorated locomotor performance. Although P5 cSTX+control mice displayed alternation between left and right limbs, P5 cSTX+vGAT mice largely failed to develop a brain-independent locomotor capacity ( Fig. 7b and Supplementary Video 3). However, we observed unilateral, non-weight-bearing reflexive steps on rare occasions (Fig. 7b, right). Therefore, we reconstructed SynGFP ON terminals to MNs and quantified their NT phenotype as a proxy to validate viral transduction efficiency. We applied PC analysis on 94 parameters and then matched the % vGAT expression at synaptic terminals to MNs for each examined limb. Reconstructed PC space segregated experimental groups based on variability related to dragging-related parameters (Fig. 7c). Furthermore, limb kinematics during treadmill locomotion broadly matched the extent of viral efficiency among experimental mice-that is, hindlimbs with low vGAT + and high vGlut2 + % synaptic inputs to MNs display ambulatory phenotype (Fig. 7d). Therefore, we conclude that acquiring vGAT accompanied by reduction of vGlut2 phenotype (Extended Data Fig. 5c,

d) by dorsal and intermediate glutamatergic interneurons deteriorates locomotor ability after neonatal injury.
Together, our results demonstrate that vGAT expression by glutamatergic interneurons in the dorsal and intermediate lamina after injury contributes to paralysis, regardless of whether it is due to spontaneous shift after adult injury or virus-mediated shift after neonatal injury (Fig. 7e).

Suppression of vGAT improves locomotor kinematics after adult injury.
Having established that defined subpopulations of vGlut2 interneurons undergo an NT phenotype switch after adult injury and that the maintenance of vGlut2 NT phenotype is critical for proficient locomotion after neonatal injury, we asked whether attenuating vGAT + expression facilitates locomotor improvement for P5 cSTX, n = 8 for P5 cSTX+control, n = 7 for P5 cSTX+vGaT). Each dot represents limb kinematics of either left or right hindlimb of one mouse. The % vGaT + is normalized to the mean of % vGaT + after adult injury (45%) and includes any limbs with SynGFP ON /vGaT + % in the range of ±2 standard deviations. Color gradient ranges from 65% to 25%, representing the highest (magenta) and the lowest (cyan) % SynGFP ON /vGaT + . PC1 scores of P5 cSTX+vGaT showed a significant difference compared to P5 cSTX and P5 cSTX+control injection (***P < 0.001, one-way aNOVa with Tukey's post hoc analysis). d, Linear regression analysis of locomotor capacity and NT composition of SynGFP ON synaptic terminals in P5 cSTX mice with control or aaV-vGaT injections. Individual PC1 values were normalized using z-score and scaled to [0,1]. Statistical analysis showed a significant deviation of the slope from zero (P < 0.001). e, Summary diagram showing a linear correlation of NT phenotype and locomotor capacity. Error bars, s.e.m. deg, degree; NS, not significant.

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after adult injury. To conditionally downregulate vGAT expression in vGlut2 interneurons, we injected floxed AAVs encoding short-hairpin RNA (shRNA) against vGAT gene expression in the lumbar segments (shRNA-vGAT-Tag; Fig. 8a). This approach allows us to express shRNA-vGAT in Cre ON neurons selectively (Extended Data Fig. 6a). We established the efficiency of this approach using a vGlut2 cre :: Tau Fig. 6b,c).
After an intraspinal injection of AAV-floxed-shRNA-vGAT in vGlut2 cre ::Tau LSL -SynGFP mice at P30, we performed a complete transection at P60 and assessed stepping capability 6 weeks after injury (Fig. 8b). All mice with shRNA-vGAT injection exhibited a few spontaneous consecutive steps, albeit limited and uncoordinated, compared to almost no steps by untreated mice (Fig. 8c,d and Extended Data Fig. 7a,b). These data suggest that the treatment influences recovery, although only partially and variably. Lack of complete recovery is not surprising as a severe injury leads to other circuit changes independent of NT phenotype switch or maintenance 15,16,[38][39][40] . Therefore, we then combined this intervention with weight-supported locomotor training known to ameliorate such maladaptive circuit-wide plasticity.
We subjected subsets of adult cSTX mice with or without shRNA-vGAT intervention to daily treadmill training of 20 minutes for 4 weeks (Fig. 8e). To train mice after adult injury, we used wide-spectrum serotonergic receptor agonists that acutely and temporally enable alternating weight-bearing stepping in rodents with complete spinal cord transection 2,3 . These agonists elevate the excitability of the spinal cord and allow training without any long-term effects 41 .
After 4 weeks of training, we quantified spontaneous locomotor capacity without serotonergic agonists. Like the shRNA-only group, the locomotor ability of the individual mouse was highly variable (Extended Data Fig. 7c). Nonetheless, PC1 captured shRNA-vGAT effects of both groups with and without training (34% of explained variance), where training further enhanced shRNA-vGAT effects ( Fig. 8f and Supplementary Video 4). Extraction and grouping of parameters highly correlated with PC1 demonstrate that shRNA-vGAT combined with daily training significantly reduced the percentage of dragging and improved joint oscillation, measures associated with locomotor recovery 41,42 (Fig. 8g, Extended Data Fig. 7d,e and Supplementary Video 4). These shRNA-treated groups exhibited a reduction and increase in immunolabeled vGAT + or vGlut2 + expression, respectively, compared to the group without any intervention after adult injury (Extended Data Fig. 8a,b). Although training alone does not improve locomotor ability after adult injury, a finding that is consistent with previous studies 2 (Fig. 8f,g), we found that training alone partially prevented NT phenotype switch-that is, fewer dorsal and intermediate excitatory interneurons expressing vGAT compared to no intervention (Extended Data Fig. 8a-c). Furthermore, training facilitated vGlut2 ON synaptic sprouting, a circuit reorganization signature that we detected after neonatal injury (Extended Data Fig. 8d), which could be a potential contributing factor to the added recovery observed in the shRNA+training group.
Together, our results demonstrate that attenuating the injuryinduced vGAT expression by excitatory neurons alone promotes locomotor pattern expression, and a combination of long-term locomotor training further facilitates distinct aspects of locomotor recovery after adult injury (Fig. 8h).

Discussion
Age-dependent plasticity of the CNS enables the spinal cord to 'learn' to walk without the brain when an injury is imposed shortly after birth. In contrast, the same injury to an adult leads to permanent paralysis. Our study reveals that age of injury-dependent locomotor circuit plasticity operates with remarkable cell type specificity, in which defined subsets of excitatory, but not inhibitory, interneurons shift the EI synaptic input ratio to MNs. Two distinct activity-dependent mechanisms, NT phenotype and synaptic sprouting, establish the age of injury-dependent, opposing pre-motor synaptic profiles with high inhibition after adult injury in contrast to increased vGlut2 synaptic input after neonatal injury. Virus-mediated opposing circuit manipulations after neonatal or adult injury suggest that the EI input ratio from pre-motor interneurons to MNs shapes locomotor ability after spinal cord injury (Fig. 8h). We discuss how these findings advance understanding of spontaneous circuit reorganization after a severe spinal cord injury, by which age of injury, cell type and activity influence the recovery process.
We found that the NT phenotype switch is one of the signatures of spinal cord plasticity, a previously unreported mechanism in the mammalian spinal cord. Studies in spinal-cord-injured cats and rodents demonstrated that modulation of inhibitory transmission is critical for improving motor output and training outcome 13,14,18,19 . These studies, however, left open the question of the source of inhibitory drive to MNs in this activity-regulated process. Unexpectedly, we found that the synaptic source of vGAT input to MN after injury was not derived from inhibitory interneurons but, rather, from vGlut2 ON interneurons switching NT phenotype.
We uncovered that a subset of spinal interneurons exhibits flexible NT phenotype, depending on the age of injury and activity level. NT phenotype switch occurs in many species, including Caenorhabditis elegans, zebrafish, Xenopus and rodents, during development and mature CNS 31,[43][44][45] . Although some neurons co-express and co-release two distinct NTs from the same synaptic terminal [46][47][48] , some release two NTs selectively from different terminals 34,35 . Our findings on spinal interneurons are consistent with the latter, in which a subset of excitatory spinal interneurons co-expresses vGlut2 and vGAT transcripts at the cell body, yet there was no accumulation of both NTs at synaptic terminals.
What is the implication of having neurons with flexible, activity-dependent NT phenotype expression in the spinal cord? We found a low level of vGlut2 and vGAT transcript co-expression at the cell body in the intact condition and a small number of vGAT + terminals in genetically labeled excitatory interneurons. This finding indicates that there are populations of neurons capable of adjusting NT expression under normal circumstances. An increase in locomotor activity leading to NT phenotype switch of defined c, Representative stick decomposition of adult cSTX mice with control (left) and shRNa-vGaT (right) injections at week 6. d, Frequency plot of stepping bouts and quantification of the maximum number of consecutive steps during a 30-second recording session (n = 6 for each group, P < 0.001 two-sided unpaired t-test). The line indicates a spline fit of the histogram distribution from the shRNa group. e, Timeline to induce stepping after adult injury with or without daily locomotor training. f, PC analysis of spontaneous stepping at week 6 (n = 6 each for no intervention, training only and shRNa-vGaT only and n = 7 for shRNa-vGaT+training). Each dot represents one mouse. Histogram plots below the PC reconstruction show differences in PC1 scores (*P < 0.05, ***P < 0.001 one-way aNOVa with Tukey's post hoc analysis). g, Examples of kinematic parameters with additive effects of daily training combined with shRNa-vGaT (n = 6 each for no intervention, training only and shRNa-vGaT only and n = 7 for shRNa-vGaT+training; *P < 0.05, **P < 0.01, ***P < 0.001 one-way aNOVa with Tukey's post hoc analysis). h, age of injury-dependent circuit signatures capture decreasing vGlut2 input due to NT re-specification of dorsal and intermediate excitatory interneurons after adult injury (1) in contrast to increasing vGlut2 pre-motor synaptic input derived from the same population to MNs after neonatal injury (2). shRNa-vGaT and training interventions respectively minimize NT phenotype switch and facilitate synaptic sprouting, therefore likely contributing to added effects of recovery when combined. Error bars, s.e.m. deg, degree; NS, not significant; Spont., spontaneous.

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midbrain neurons in intact mice 23 suggests that motor activity may also modulate the baseline level of NT phenotypes in the intact spinal cord, like for motor learning. Likewise, such flexibility offers an opportunity to harness NT phenotype switches as a therapeutic approach to augment rehabilitative training and prevent maladaptive circuit functions after a severe injury 15

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To date, mechanisms that underlie the closure of the enhanced plasticity time window after neonatal injury remain unknown. The sensitive period for achieving proficient locomotion without the brain closes around P15 in rodents 49 , a timepoint when a severe injury leads to a diverging outcome of proficient locomotion or paralysis. This time window corresponds to when corticospinal inputs reach the lumbar spinal cord, and brainstem-descending pathways establish mature connectivity patterns [50][51][52] . It is, therefore, possible that cell-intrinsic plasticity of spinal interneurons changes fundamentally around this time to facilitate the integration of descending inputs and local circuits to enable the execution of proficient motor programs. Our work revealed that baseline PA activity is necessary to maintain glutamatergic NT phenotype by excitatory interneurons after neonatal injury, which we demonstrate to be an essential mechanism to learn to walk without the brain. However, PAs do not regulate gene expression of NTs after adult injury in a similar manner. Instead, spinal circuits injured as an adult require intense, movement-directed somatosensory activation to partially prevent the gain of vGAT phenotype by glutamatergic interneurons and for synaptic sprouting. In support of this idea that cell-intrinsic plasticity changes around P15, the end of the sensitive period for the age of injury-dependent locomotor circuit plasticity coincides with a marked shift in gene expression pattern throughout the CNS 53 .
We also found that subpopulations of excitatory interneurons and their synaptic connectivity profiles define locomotor capacity after complete spinal cord injury. In contrast, NT phenotype and connectivity from the inhibitory interneurons to MNs are hardwired and unaffected by injury or age of injury. Of the three excitatory interneuron populations examined, dorsal and intermediately located interneurons were much more plastic than ventral interneurons. Based on findings from PA ablation, activity-dependent NT phenotype re-specification and sprouting, we speculate that spatial positioning of these neurons-that is, their access to PA input-is a critical factor in the observed cell-type-specific plasticity. These dI3/5 and V2 interneurons reside along the dorsoventral and mediolateral axes with abundant PA innervation, whereas most V3 neurons sit medioventrally where PAs innervate sparsely. Recent transcriptomic analysis revealed cell-type-specific response to injury along the dorsoventral axis 54 . These findings point to the possible importance of spatial positioning within the spinal cord.
Consequently, the identity and functional roles of more plastic excitatory interneurons in locomotion, per se, may be a fortuitous secondary factor to facilitate locomotion without the brain. For instance, defined subpopulations of V2 neurons are responsible for either locomotor rhythm or patterns with abundant interconnections within each functional group 55,56 . In contrast, the roles of dI3 and dI5 interneurons in locomotion are unknown. Tlx3 is a selector gene that determines a glutamatergic over GABAergic neuron cell fate during development 57 . NT phenotype switch in the Tlx3 population is likely unrelated to the function of Tlx3 as we study them in adulthood when Tlx3 is expressed at a low level. Nonetheless, our work suggests that the vGlut2 synaptic drive from these neurons to MNs is critical for regulating locomotion without the brain. Roles of V3 interneurons are yet to be defined.
The age of injury-dependent EI input balance from spinal interneurons is consistent with the need for increased excitability for movement generation after injury [12][13][14] . However, the finding that benefits locomotor recovery may seem paradoxical to maladaptive hyperexcitability observed after spinal cord injury 15,16,40 . Therefore, identifying other sources of MN excitation and the extent of their contribution to recovery and maladaptation remain essential. In addition to pre-motor neurons, MN physiology after a spinal cord injury represents a collective shift in excitability of MN 15,16 and vGlut1 ON input of muscle spindle Ia afferents and their regulation by GABApre neurons. They are known culprits to mediate maladaptive state of hyperexcitability leading to muscle spasticity [58][59][60][61][62] . It remains unclear whether and how distinct local circuit modules interact to achieve the coordinated activation and inhibition of the local circuits in movement-matched sequence and vigor after neonatal injury or shRNA-treated mice after adult injury.
In summary, we identified that NT phenotype and synaptic sprouting of specific populations of excitatory interneurons serve as a neuronal basis of age of injury-dependent locomotor capacity after severe spinal cord injury. This finding stresses the importance of targeting defined neurons in rehabilitative strategies after spinal cord injury. Together with recent studies in humans demonstrating benefits of increased excitability of the spinal cord temporarily and acutely with electrical stimulation [4][5][6] and rodent models to modulate EI balance of circuits 42,63 , enacting optimal EI balance below lesion may be achieved in part by regulation of interneurons. Similar concepts may apply to other traumatic CNS disorders that highly rely on plasticity attributed to the age of patients and circuit-specific targeting to regulate EI balance for locomotor recovery.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41593-022-01067-9.  29 ) mouse strains were maintained on a mixed genetic background (129/C57Bl6). Adult mice of both sexes, aged 2-6 months, were used for all experiments. Mice were group housed, and all mice were kept on a 12-hour light/dark cycle in a facility where temperature was kept at 22 ± 2 °C and ~50% humidity. All animal procedures were conducted in accordance with Belgian regulations and were approved by the institutional ethical committee of KU Leuven.

NaTurE NEuroScIENcE
Spinal cord injury and daily care. For neonatal complete spinal cord transection, 5-day-old pups were anesthetized with ice, and muscles were separated along the midline to expose the vertebral column. Laminectomy was performed using forceps at spinal segment ∼T10, and the spinal cord was completely transected using micro-scissors. After surgery, mice were placed in a heat chamber until fully awake. All other surgical procedures were performed under aseptic conditions and full general anesthesia with isoflurane in oxygen-enriched air (1-2%). Adult mice underwent a mid-dorsal skin incision to access the spinal cord, and a laminectomy was made over the spinal segment T10. A complete spinal cord transection was performed using micro-scissors. Analgesia (Metacam, Boehringer Ingelheim, 0.5 mg ml −1 ) was provided after surgery, and antibiotics (Cefazoline, Sandoz, 5 mg kg −1 ) were provided for 3 days after surgery. Mice were housed on a 12-hour light/dark cycle with ad libitum access to food and water and cared for twice daily with manual bladder voiding. Complete transections were confirmed postmortem by visual inspection of the spinal cord.
Kinematic training and recordings. Bipedal locomotor kinematic recordings were performed using the high-speed motion capture system Vicon (Vicon Motion Systems). Ten infrared cameras (200 Hz) were used to detect 3-mm reflective markers attached bilaterally on the mice overlying the iliac crest, the greater trochanter (hip), the lateral condyle (knee), the malleolus (ankle) and the base of the metatarsal phalangeal joint. Positions of the markers were reconstructed offline in three dimensions (3D) using Vicon Nexus software (version 2.12). The hindlimb was modeled as interconnected segments, and joint angles were calculated accordingly. Parameters (Supplementary Table 1) describing gait timing, joint kinematics, limb endpoint trajectory and stability were computed for each gait cycle using custom-written MATLAB scripts and according to methods detailed previously 28 . Locomotor training started 2 weeks after adult spinal transection and occurred for 20 minutes per day for 4 weeks on a motorized treadmill with body weight support (Robomedica) at 5-15 cm s −1 , after intraperitoneal injection of monoaminergic agonists, consisting of 5-HT1A/7 agonist 8-Hydroxy-DPAT hydrobromide (Sigma-Aldrich, H140, 0.375 mg kg −1 ) and 5-HT2A/C agonist Quipazine (Sigma-Aldrich, Q1004, 0.125 mg kg −1 ). Neonatally injured or adult injured mice with muscle atrophy, incomplete spinal cord injury or occasional spinal cord degeneration were excluded from the study.

Kinematic analysis.
Step 1: Locomotor patterns are recorded using high-resolution cameras.
Step 2: Custom-written MATLAB scripts are used to reconstruct kinematic parameters. All variables computed for each task are specified in Supplementary  Table 1. Approximately 15-20 steps were extracted per mouse.
Step 3: All computed variables were averaged for each mouse independently. All body-size-dependent parameters were normalized according to body weight. The matrix combining all mean values of variables from all mice of analysis was then subjected to a PC analysis. For this purpose, we used the correlation method, which adjusts the mean of the data to 0 and the standard deviation to 1. A new set of synthetic uncorrelated variables-that is, the PCs-each explains the maximum possible amount of variance.
Step 4: The new coordinates of gait patterns along each PC, termed PC scores, are extracted for each mouse. PC scores represent gait patterns in the 'denoised' PC space to visualize differences between experimental conditions.
Step 5: PC scores are averaged for each experimental condition and represented in histogram plots to identify the type of information differentiated along each PC axis.
Step 6: Each PC is a linear combination of the original parameters with appropriate weights, termed 'factor loadings' . The values of factor loadings range from −1 to 1 and correspond to correlations between original parameters and a given PC.
Step 7: Factor loadings with a high value (|factor loading| > 0.5) are extracted, color-coded based on their correlation value and regrouped into functional clusters.
Locomotor analysis after shRNA injection. For the shRNA+training group, mice underwent daily locomotor training starting from 2 weeks after lesion for 4 weeks. Kinematic recordings were performed at week 5 and week 6 for all four groups (no intervention, training only, shRNA only and shRNA+training). The number of consecutive steps, dorsal and plantar steps combined and bouts were quantified over one recording session (30 seconds) from either week 5 or week 6. Dragging is defined as a contact of the dorsal surface of the foot to the ground during the swing phase. After kinematic reconstruction, each step cycle is manually inspected by an experimenter with 100-Hz digital camera recordings to confirm foot ON/OFF segments. DTX delivery for PA ablation. DTX (Sigma-Aldrich, D0564) was injected intraperitoneally for adult mice (100 mg kg −1 ) and subcutaneously for P5 mice (100 µg kg −1 ) following an established protocol 30 . To verify ablation efficiency, we determined vGlut1 ON afferent density postmortem. Inclusion criteria were set at >50% ablation after normalization compared to intact conditions. Virus production. G-deleted rabies with fluorescent Tag was amplified and purified from local viral stocks following established protocols 66 . All AAVs used in this study were of genomic titers >1 × 10 13 .

Intraspinal injections.
Under deep general anesthesia using either isoflurane enriched with oxygen (1-2%) for adult mice or hypothermia for P5 mice, a mid-dorsal skin incision and laminectomy were performed to expose the spinal cord. The intraspinal injection was made using a pulled calibrated glass pipette (World Precision Instruments) by multiple short pulses (3 ms, 0.5 Hz) using a picospritzer (Parker Hannifin). Adult injured mice. A bilateral intraspinal virus injection of AAV-floxed-shRNA-vGAT-Tag was performed in lumbar segments of ~3-week-old vGlut2 cre :: Tau LSL-lacZ-SynGFP-INLA mice, 2 weeks before a T10 complete spinal transection. After 2 weeks of recovery, maximum consecutive steps were counted over one recording session (30 seconds) at week 5 or week 6. For the shRNA+training group, mice underwent daily locomotor training for 4 weeks, and kinematic recordings were obtained. Virus infection efficiency was determined postmortem, with inclusion criteria set at <30% mean expression of vGAT + at SynGFP ON terminals. A subset of mice underwent a mock injection before the injury as a control.
Descending circuit tracing with G-deleted rabies. Using isoflurane anesthesia, mice underwent laminectomy to expose the surface of the spinal cord. A pulled calibrated glass pipette (Drummond Scientific) was used for local application of 100 nl of virus by multiple short pulses (3 ms, 0.5 Hz) using a picospritzer (Parker Hannifin). After surgery, mice were administered Metacam (5 mg kg −1 ) subcutaneously for analgesia. Mice were perfused for analysis 4 days after injections.
Histological analysis. We used spinal cord tissue >1 month after injury for both P5 and adult cSTX groups. To make sure that age of tissue per se is not the driving factor for vGlut2/vGAT gene expression and synaptic profile connectivity, we confirmed that quantification obtained from P5 cSTX tissue at an older age (that is, age-matched to adult cSTX at ~P75-90) does not differ from P5 cSTX at ~P35.

March 2021
Corresponding author(s): Aya Takeoka Last updated by author(s): Mar 23, 2022 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

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