Spinal cord injury (SCI) is often a permanent debilitating disorder that disrupts sensory and motor function below the level of injury. Damage to sensorimotor pathways after SCI causes changes in neural circuitry, acutely and chronically. Behavioral improvements are often marked by the strengthening and rewiring of damaged and spared connections (Waters et al., 1996; Burns et al., 1997; Bilchak et al., 2021). Recent advances in treatments for SCI have demonstrated potential for promoting recovery after injury. In particular, epidural electrical stimulation (EES) applied to the lumbosacral spinal cord mediates functional improvements in patients with chronic SCI (Herman et al., 2002; Courtine et al., 2009; Harkema et al., 2011; Karimi et al., 2013; Angeli et al., 2014; Crosbie et al., 2014; Possover, 2014; Grahn et al., 2017; Formento et al., 2018; Gill et al., 2018). EES works by recruiting large diameter peripheral afferents, such as group I and II proprioceptive afferents and group II cutaneous afferents, that enter through the dorsal roots (Bouyer and Rossignol, 1998; Rossignol et al., 2006; Capogrosso et al., 2016). Activation of these afferents is thought to modulate synaptic connections that ultimately drive agonist/antagonist muscle recruitment. Precisely which pathways are responsible is an open question; it may be relatively localized to the spinal cord, or it may be indirect; relying on sensory information sent to supraspinal centers for motor correction and learning (Eisdorfer et al., 2020). Thus, mapping the circuitry that EES influences is a critical goal for the field (Spataro et al., 2005; Thelin et al., 2011; Côt et al., 2017), with clinical import.

While the effects of electrical stimulation can be inferred with histological methods, it is difficult to know precisely which neurons have been stimulated. This makes pinning down pathways of plasticity harder. Genetically encoded tools make this possible: by fusing the tool with a fluorescent protein, neurons that are manipulated are also labeled (Haery et al., 2019). This motivated us to use the genetically encoded excitatory DREADDs receptor hM3Dq in an EES-like context. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) are engineered “chemogenetic” receptors that enable targeted neural modulation in freely behaving animals through selective binding of a ligand, in this case clozapine-N-oxide (CNO) (Roth, 2016). DREADDs expression was driven by adeno-associated virus (AAV), and due to the coupled fluorescent protein, facilitate characterization of targeted neurons and the second-order neurons they influence (Wu et al., 2020).

The aims of this study were 2-fold: (1) to determine whether activation of large diameter peripheral afferents with hM3Dq DREADDs influences recovery from a hemisection spinal cord injury in the rat model and (2) to begin to uncover any underlying mechanisms of plasticity. Connections between afferents and motor pools or interneuronal networks may have important implications for functional recovery after injury. In particular, afferent activation impinging upon motor pools, either monosynaptically or polysynaptically, could increase appropriate activation/inactivation of agonist/antagonist muscle groups during training and rehabilitation (Hultborn et al., 1971; Mears and Frank, 1997; Dimitrijevic et al., 1998; Guertin, 2012). Furthermore, afferent information relayed to supraspinal centers may contribute to increased coordination of movement, motor learning, and motor correction after injury (Brownstone et al., 2015; Bui et al., 2016; Fink and Cafferty, 2016; Côt et al., 2017; Kim et al., 2017; Asboth et al., 2018; Eisdorfer et al., 2020; Gao et al., 2021). For example, interneuronal networks within Clarke's column (nucleus dorsalis or Clarke's nucleus), located within the thoracic and lumbar spinal segments, contain dorsal spinalocerebellar (dSC) tract neurons that can relay proprioceptive sensory signaling from the hindlimb to cortical motor centers (Kim et al., 1986; Aoyama et al., 1988; Edgley and Gallimore, 1988; Bosco et al., 2000; Bosco and Poppele, 2003; Hantman and Jessell, 2010; Sengul and Watson, 2012). As such, sprouting and synaptogenesis within lamina that contain motor pools and Clarke's column could support recovery after SCI.

To achieve these aims, we expressed hM3Dq DREADDs in large diameter peripheral afferents innervating the lumbar spinal cord to gain an understanding of the influence of selective continuous activation of large-diameter afferents can have on the hindlimb after SCI. In animals expressing DREADDs, we report higher densities of fluorescent axons in the motor pools and Clarke's column of the lumbar spinal cord, which may indicate that increased activation of afferents by DREADDs resulted in increased afferent sprouting and synaptogenesis onto interneurons and motorneurons. Analyses of kinematics for five points on the hindlimb—anterior superior iliac spine (ASIS), greater trochanter (hip), knee, ankle, and metatarsophalangeal (MTP) joints—revealed that chronic DREADDs activation leads to increased height of the hindquarters (e.g., indicated by the ASIS and hip heights), putatively indicating increased motorneuron activation and muscle recruitment. DREADDs animals also display ankle joint angles that are closer to the pre-injury condition. This may suggest that increased afferent activation helps to promote appropriate ankle movements during the step cycle. Interestingly, animals with DREADDs exhibit a buckling phenomenon, or a collapse of the hindquarters, in the absence of DREADDs activation (e.g., by withholding CNO administration) as observed in a larger range of ASIS heights during treadmill locomotion. Future work, such as using cFOS to examine and map changes in interneuronal networks, could seek to more directly tie changes in kinematics to observed changes in plasticity. Furthermore, these data that activate both proprioceptive and exteroceptive afferents form a baseline data set against which further work that restricts expression to proprioceptive or other subsets of afferents can be compared.

Animals were injected with hM3Dq DREADDs or control virus in lumbar (L2-L5) DRG and received a right-side T9 hemisection SCI (Figure 1A). Figures 1B–D present illustrative schematics of hindlimb kinematics acquisition and analysis. Five hindlimb joints/points were labeled: ASIS (anterior superior iliac spine), hip, knee, ankle, and MTP (metatarsophalangeal). Recordings were obtained at 250 FPS during treadmill locomotion at 5 speeds (16, 20, 24, 28, and 32 cm/s). Estimation of joint positions were extracted with DeepLabCut (Mathis et al., 2018; Nath et al., 2019) and then reconstructed in 3D (Maghsoudi et al., 2019). An experimental timeline is presented in Figure 1E: kinematics were recorded pre-injury, and 1, 2, 4, and 6 weeks post-injury; and treadmill training was conducted thrice weekly.

Advances in therapeutic interventions for individuals with SCI, such as epidural electrical stimulation (EES), have demonstrated the ability to mediate functional improvements putatively via the rewiring damaged and spared circuitry. Uncovering the neural circuit changes involved in promoting functional recovery is a critical challenge for the field, and while electrical spinal cord stimulation modalities are robust, modern genetic tools offer advantages in deterministically tracing affected circuits and quantifying plasticity. In this study, we sought to uncover some of the mechanisms that may underlie rehabilitation with excitation of large diameter afferents, and describe concomitant changes in locomotion with detailed kinematics. With our transduction viral approach, we coupled hM3Dq (excitatory) DREADDs expression with a fluorescent protein for immunohistological characterization of neural circuit changes involved with activation of targeted lumbar afferents.

It is well-accepted that synaptic plasticity plays an important role in behavioral improvements after SCI (Waters et al., 1996; Burns et al., 1997). In this study, we report larger densities of mCherry fluorescence in the DREADDs group in both the motor pools and Clarke's column (Figure 2). Given that we observe similar spared tissue at the epicenter of the hemisection (Kloos et al., 2005) and AAV2 transduction rates in the DRG of both groups, our results suggest that DREADDs activation may have induced plasticity of targeted afferents onto interneurons and motorneurons.

Axonal sprouting and synaptogenesis within motor pools can directly and indirectly drive muscle activity by supplementing motorneuron activation after SCI. Directly, plasticity within monosynaptic connections of group Ia afferents onto motorneurons that occur within the same muscle target could be influential in regaining of volitional control of affected muscles (Eccles et al., 1957; Takeoka et al., 2014; Moraud et al., 2016; Takeoka, 2019; Takeoka and Arber, 2019; Eisdorfer et al., 2020). Plasticity in the lumbar spinal cord promoted by hM3Dq DREADDs in multiple muscles within the same extensor or flexor group may support an increase in overall muscle force generated as indicated by the lifted and potentially more stable hindquarters seen in Figure 3. The evidence suggests that chronic DREADDs activation of afferents is inducing behavioral differences, potentially mediated by the plasticity observed in histology. DREADDs activation may cause stiffer hindlimb musculature by co-activating extensors and flexors. It may also extend the limb by activating more extensors than flexors, or simply because extensor musculature and/or moment arms are larger (Latash, 2018). With stiffer muscles and a potential bias toward extension, DREADDs animals may be capable of lifting their hindquarters higher than their control counterparts. Neuromechanical models of afferent excitation in moving rodents have suggested that increasing afferent excitation results in a more extended limb (S. Danner, personal communication; Danner et al., 2017). Interestingly, DREADDs animals exhibit a more flexed ankle joint angle during the transition between stance to swing (Figure 4A) and during the swing phase (Figure 4B) at intermediate time points. Afferent activation by DREADDs may promote more appropriate ankle joint movements during the end of the stance phase, aiding in push-off and reducing toe-drags upon liftoff.

Indirectly, plasticity may also occur where modulated afferents synapse onto interneuronal circuitry that conveys information to the motor pools, such as interneurons involved in lumbar central pattern generators (CPGs). Plasticity onto CPG circuitry may aid in adapting to perturbations and transmission of rhythmic activity for hindlimb coordination after injury (Cowley et al., 2008; Bui et al., 2013, 2016; Young, 2015; Danner et al., 2017; Laliberte et al., 2019; Shepard et al., 2021; Zholudeva et al., 2021), which may underlie observed step durations that are closer to the pre-injury condition in DREADDs animals as compared to their control counterparts at intermediate times (Figure 3I). In theory, activation by hM3Dq DREADDs in muscles about the hip could serve to promote hindlimb coordination by helping to control phase transitions and entrain rhythmic flexor and extensor activation (Andersson and Grillner, 1983; Kriellaars et al., 1994; Hiebert et al., 1996; Kiehn, 2006; Onushko, 2009). This may seem counterintuitive given the tonic nature of DREADDs activation, but tonic EES is thought to promote phase-appropriate changes in muscle activity, for appropriate levels of stimulation, due in part to the filtering properties of the CPG circuitry (Moraud et al., 2016). If true, afferents expressing hM3Dq DREADDs may be influential in the oscillating activation of neurons located within lumbar CPGs. Further studies could more directly tie this observation by gathering information from muscle recordings during locomotion bouts.

The aforementioned kinematic variables that were significantly different in DREADDs activated animals at intermediate time points, and then regressed to match the controls, could reflect plasticity and motor learning at these time points to handle the nature of increased afferent excitability (Figure 3F, variation in ASIS height, week four; Figure 4B, mean ankle angle during swing, week two). The divergence of controls and DREADDs animals in weeks four to six (Figures 3A,F,G, 4D,F) likely reflect the critical transitory phase where plasticity can be more strongly influenced, after which changes are more difficult to induce. During this phase, the nervous system is likely learning to adapt to the increased excitability of the afferents, attempting to make this input functional. After increased variability in ASIS height in week 4 (Figure 3F), ASIS height is stabilized closer to baseline (Figure 3A). For ankle angle, however, increased excitability pushes it further from baseline, to a more flexed angle, before recovery brings it back in line with controls (Figure 4B). The nature of increased excitability with DREADDs comes into play here; whether it tonically activates neurons or leads to more extended bursts of activity after a phasic activation, depending on dose, may influence this transition period and is an interesting point for future study, and for comparison with phase-locked methods of stimulation.

New and strengthened synapses in Clarke's column by hM3Dq DREADDs activation may have implications for relaying sensory information to supraspinal centers during locomotion. Under normal conditions, the dorsal spinalocerebellar (dSC) tract neurons within Clarke's column relay sensory information to cortical motor centers for error adjustments and motor learning. Thus, in the injured condition, plasticity of targeted afferents, including groups Ia and Ib proprioceptors as well as group II cutaneous afferents, could plausibly more efficiently relay information about hindlimb position and movement to dorsal spinalocerebellar (dSC) tract neurons (Kim et al., 1986; Aoyama et al., 1988; Edgley and Gallimore, 1988; Bosco et al., 2000; Bosco and Poppele, 2003; Hantman and Jessell, 2010; Sengul and Watson, 2012). Future studies that record brain activity may help to unearth the effects of increased activation of peripheral afferents on supraspinal centers, particularly those involved in motor learning and error correction. Importantly, upregulation of large diameter peripheral afferents by DREADDs may be working similarly to activation by electrical stimulation. A hallmark of the EES paradigm is its ability to promote plasticity and modulate neural circuitry for use in locomotor tasks (Harkema et al., 2011). Higher order neural centers could filter uniformly boosted afferent input to use these enhanced sensory cues appropriately. Circuitry that is capable of siphoning general hindlimb-mediated afferent upregulation may also reside within lumbar CPGs or other spinal processing centers (Capogrosso et al., 2013). Like EES, afferent activity elevated by DREADDs activation may also be interpreted by neural circuitry appropriately.

In our study, we observed that joint angle variation (here computed as the standard deviation of the joint angle time series for a stride; so a measure of range of motion of the joint angle) within the DREADDs group was more pronounced in angles of the hip joint (Figure 4F) and smaller in the ankle joint (Figures 4C,D). These differences across joints could reflect differences in activation of muscles given the innervation of the DRGs that we transduced; or it could reflect differences in muscle mass about the different joints. Motor noise is multiplicative (Valero-Cuevas et al., 2009); therefore if we had injected equal amounts of additional activation across the motor pools, the larger muscles about the hip could inject more noise than smaller muscles about the ankle, for a given level of activation, resulting in larger ranges of motion; however it may still be expected that both hip and ankle would be larger in DREADDs than controls.

Elimination of large diameter peripheral afferent activation after SCI has been demonstrated to induce significant kinematic changes. For example, sensorimotor improvements reported with EES are enabled when stimulation is turned on and tuned to a specific frequency and amplitude (Harkema et al., 2011; Capogrosso et al., 2013; Angeli et al., 2014; Formento et al., 2018). Additionally, studies with proprioceptive ablation following recovery from SCI permanently reverts improvements in functional recovery to the injured state (Takeoka et al., 2014; Takeoka, 2019; Takeoka and Arber, 2019). Our results corroborate these findings. In our study, we report that withdrawal of DREADDs agonist CNO results in a grossly observed “buckling” or “collapse” event of the hindquarters during treadmill locomotion, a phenomenon reported in mice with spinal cord injury (Basso et al., 2006). We qualitatively define the buckling phenotype as follows: DREADDs animals would take one to several normal strides; suddenly drop their hindquarters onto the treadmill belt and remain seated on the belt moving their hindlimb as if they were mid-stride, but unable to stand; correct their error by lifting their hindquarters off the belt; and begin locomoting normally again (Figure 5). The range of overall hindquarter height (see ASIS height) in the absence of hM3Dq DREADDs activation was larger due to the buckling event. It is interesting to speculate about the source of this phenotype. One simple interpretation is that adaptations have occurred to perform an activity (e.g., locomotion) with a pattern of peripheral afferent activity induced by DREADDs activation. As such, the neural circuitry might be forced to compensate differently in the absence of this pattern of activity. Another interpretation is that the lack of DREADDs activation generates less stiff hindlimb musculature, which in turn results in the collapsing of the hindquarters (Latash, 2018). Furthermore, weight support mechanisms are potentially driving the buckling event. With DREADDs activation, afferent input may be sufficient to avoid buckling events. By extension, in the absence of DREADDs activation, there may be insufficient afferent feedback to prevent loss of weight support (Norton and Mushahwar, 2010; De Leon and Dy, 2017). Finally, the absence of DREADDs activation in cutaneous afferents in the footpad may have implications on shifts the body produces to compensate for the lack of expected tactile information, resulting in the buckling event (Park et al., 2019). Results suggest that DREADDs activation is helpful, but perhaps not required, to access newly formed and strengthened pathways since DREADDs animals demonstrate the ability to lift their hindquarters up off the treadmill belt (e.g., the termination of the buckling event). This may indicate there are underlying circuit changes that are accessible for motor correction even in the absence of DREADDs activation. As such, continued exercise training in the absence of DREADDs activation may eliminate the buckling phenotype altogether. It is entirely possible that the combination of DREADDs activation and neural circuit plasticity are required to prevent a buckling event from occurring.

Outside of the buckling phenomenon, we did not observe dramatic changes in function with withdrawal of DREADDs activation in week 7 (Figures 3, 4: compare weeks 6, 7, and 8; including orange and blue shaded weeks). Features in DREADDs animals that had risen above or below controls stayed there (above: Figures 3A–E,G,H, 4F; below: Figures 3F, 4D). This could indicate that underlying plasticity has “taken over” in supporting the changes we observe in kinematics in later weeks, and that DREADDs activation is no longer necessary.

Sensorimotor improvements after SCI are often marked by plasticity of damaged and spared neural circuitry. In this study, we demonstrate the use of hM3Dq (excitatory) DREADDs in lumbar large diameter peripheral afferents in a SCI model, similar to epidural electrical stimulation of the lumbosacral spinal cord. Unlike electrical stimulation, genetic techniques enable for characterization of neural pathway changes that occur with enhanced afferent feedback, such as our observation of increased plasticity within motor pools and Clarke's column in animals with DREADDs. This plasticity might underlie kinematic differences between DREADDs animals and controls that we observe, such as increased height of the hindquarters as well as more appropriate ankle joint movements during the step cycle. Future studies could further trace the mechanisms of plasticity and utilize muscle recordings to illuminate where DREADDs is most influential in inducing biomechanical changes.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Temple University Institutional Animal Care and Use Committee.

JE, ML, GS, and AS contributed to conception and design of the study. JE, HS-B, SS, JC, TC, RS, GM, BR, and AS performed surgeries and experiments, collected data, and contributed to figure generation. JE and AS performed the statistical analysis and wrote sections of the manuscript. JE, RS, ML, GM, and AS contributed to revision of the manuscript. All authors read and approved the submitted version.

This work was supported by NIH NINDS grant 1R01NS114007-01A1 to AS, NIH NINDS grant R01NS117749-01 to GS, and NIH/NINDS grant NS114007 to ML, Shriners Hospitals for Children Grant #85115, Craig H. Neilsen Foundation Senior Research Grant (#546798) to AS, Craig H. Neilsen Foundation Project #598563 to ML, and the Shriners Viral Core Grant #84051-PHI-21 to GS.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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