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. 2021 Sep 9;10:e65304. doi: 10.7554/eLife.65304

The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord

Yunuen Moreno-Lopez 1,†,, Charlotte Bichara 1,†,§, Gilles Delbecq 1, Philippe Isope 1, Matilde Cordero-Erausquin 1,
Editors: Muriel Thoby-Brisson2, Ronald L Calabrese3
PMCID: PMC8439650  PMID: 34497004

Abstract

It is generally assumed that the main function of the corticospinal tract (CST) is to convey motor commands to bulbar or spinal motoneurons. Yet the CST has also been shown to modulate sensory signals at their entry point in the spinal cord through primary afferent depolarization (PAD). By sequentially investigating different routes of corticofugal pathways through electrophysiological recordings and an intersectional viral strategy, we here demonstrate that motor and sensory modulation commands in mice belong to segregated paths within the CST. Sensory modulation is executed exclusively by the CST via a population of lumbar interneurons located in the deep dorsal horn. In contrast, the cortex conveys the motor command via a relay in the upper spinal cord or supraspinal motor centers. At lumbar level, the main role of the CST is thus the modulation of sensory inputs, which is an essential component of the selective tuning of sensory feedback used to ensure well-coordinated and skilled movement.

Research organism: Mouse

Introduction

Several brain and spinal structures have been involved in the generation and control of movement in a task-dependent manner. The primary motor cortex is classically associated to forelimb skilled movements (Starkey et al., 2005; Guo et al., 2015; Miri et al., 2017; Galiñanes et al., 2018) and to complex locomotor tasks (obstacles crossing, leaning scale or beam, etc.; Beloozerova and Sirota, 1993; DiGiovanna et al., 2016; Wang et al., 2017; Bieler et al., 2018) while its role in rythmic locomotion appears, if anything, minor (Beloozerova and Sirota, 1993; DiGiovanna et al., 2016). A coordinated movement relies not only on a proper motor command, but also on the online analysis of an ongoing multisensory feedback indicating whether the movement has reached the planned objective or requires correction (Akay et al., 2014; Fink et al., 2014; Azim and Seki, 2019). To optimize the analysis of the overwhelming quantity of reafferent information, specific tuning of peripheral feedback is possible through inhibition of sensory information at different stages of the sensory pathway, a phenomenon known as ‘sensory gating’ (Seki and Fetz, 2012; McComas, 2016).

At the spinal cord level, sensory gating can be mediated by presynaptic inhibition of sensory inputs through primary afferent depolarization (PAD), a powerful mechanism controlled both by supraspinal centers and by adjacent primary afferents (Eccles et al., 1962). While multiple mechanisms have been demonstrated (Shreckengost et al., 2021), the most studied cause of PAD is presynaptic GABA-A-mediated depolarization. Primary afferents have indeed a high chloride content and respond to GABA by a depolarization that has been shown to reduce the amplitude of incoming action potentials and thus inhibit synaptic transmission (Prescott and De Koninck, 2003; Doyon et al., 2011). PAD has been involved in the adjustment of reflex excitability (Rudomin and Schmidt, 1999), and its absence results in a pathological context associated with spasticity (Caron et al., 2020). Proprioceptive feedback is required for skilled locomotion (Akay et al., 2014), and presynaptic inhibition of sensory feedback is essential for performing smooth forelimb skilled movements in mice (Akay et al., 2014; Fink et al., 2014). Interestingly, PAD can also be evoked by stimulation of the sensorimotor cortex in primates, cat, and rats (Andersen et al., 1962; Carpenter et al., 1963; Andersen et al., 1964; Abdelmoumène et al., 1970; Eguibar et al., 1994; Wall and Lidierth, 1997). This cortically evoked PAD may underlie cortical sensory gating at the spinal level and contribute to the cortical involvement in voluntary movements, in addition to the classical drive of motoneurons. In this study, we aimed at elucidating the neuronal pathways conveying the cortical motor command and sensory control to the hindlimb in mice. This cortical control of the spinal cord, either to trigger motoneurons or to modulate sensory information, can rely on multiple pathways, the corticospinal tract (CST) being the most direct one. Its involvement in motor control is long acknowledged, although its precise contribution might vary depending on the motor task (locomotion vs. skilled movement) or targeted limb (forelimb vs. hindlimb) (Miri et al., 2017; Wang et al., 2017; Karadimas et al., 2020). The CST has also been involved in cortically evoked PAD in cats as a pyramidotomy abolishes it (Rudomin et al., 1986). However, discriminating sensory vs. motor contribution has been hampered by the lack of proper tools to segregate functionally or anatomically overlapping paths.

Importantly, none of the CST functions can rely exclusively on direct CST contacts onto motoneurons (MN) as these represent the exception rather than the rule. Indeed, monosynaptic CST-MN contacts are present only in some higher primates, while CST-interneuronal connections are preponderant (in adults) across species (rodents and primates) (Alstermark et al., 2011; Ebbesen and Brecht, 2017; Ueno et al., 2018). In the recent years, specific genetic factors have enabled the identification of several interneuronal populations, and of their inputs, including those from the cortex (Hantman and Jessell, 2010; Bourane et al., 2015; Abraira et al., 2017; Ueno et al., 2018). But neurons with identical genetic markers do not necessary share identical function: in these genetically distinct populations, up to 65% of the neurons receive CST inputs (Ueno et al., 2018), while there is a significant fraction of neurons that is not targeted by the CST. Although manipulating these populations has been extremely informative to better understand their role, it remains unclear whether the changes observed upon these manipulations can be specifically attributed to the neuronal subset that receives inputs from the CST or to the subset that does not. Therefore, it is still difficult to assess the specific role of the targets of the CST in movements.

In this study, we have investigated the corticofugal paths conveying motor command and modulation of sensory inputs through PAD. We have in particular interrogated the contribution of the CST and its direct lumbar targets through the development of an innovative intersectional viral strategy. We show that cortically evoked PAD in lumbar primary afferents is conveyed exclusively by the CST through a population of lumbar interneurons. In contrast, our results suggest that motor command to the hindlimbs is relayed either through supraspinal motor centers, or through the CST with a propriospinal relay in the upper cord. The role of the lumbar CST thus appears to be mainly the modulation of sensory inputs, which may in turn selectively regulate sensory gain involved in the refinement of motor control during movement.

Results

PAD and muscular contractions originate in the same cortical area

The rodent sensorimotor cortex has a highly somatotopic organization (Ayling et al., 2009). Mainly studied in a motor perspective, it has been repeatedly mapped for its ability to provoke motor contraction (Li and Waters, 1991; Ayling et al., 2009), but rarely for its ability to induce PAD (Wall and Lidierth, 1997). Cortically evoked PAD may inhibit the transmission of sensory information from primary afferents to the central nervous system and can be experimentally assessed by recording dorsal root potentials (DRPs, Figure 1A). We thus recorded DRPs in vivo in lumbar roots L4–L6 (conveying hindlimb sensory inputs) of Thy1-ChR2 mice, expressing channelrhodopsin2 in most neuronal cells including layer V cortical neurons (Arenkiel et al., 2007). DRP recordings were performed in isoflurane-anesthetized mice, and trains of photostimulations (5 × 8 ms pulses, 1 ms apart) were applied to activate ChR2 at different positions of the surface of the sensorimotor cortex according to a 500-µm-spaced grid. The area inducing the DRPs was centered at AP = −0.75 mm, L = 1.5 mm (Figure 1C, n = 7 mice). Similar photostimulation of this area in mice expressing only the fluorescent reporter EGFP in corticospinal (CS) neurons leads to no DRP signal (see below and Figure 3—figure supplement 1). On a different group of animals, electromyographic (EMG) recordings were performed in the tibialis anterior (TA) under ketamine/xylazine anesthesia because EMGs cannot be evoked from cortical stimulation under isoflurane anesthesia (see Materials and methods). The sensorimotor cortex was similarly mapped with trains of photostimulations (6 × 1 ms pulses, 2 ms apart) and the area inducing EMG was centered at AP = −0.75 mm, L = 1.75 mm (n = 5 mice; Figure 1C). Again, similar photostimulation of this area in mice expressing only EGFP in CS neurons leads to no EMG signal (see below and Figure 3—figure supplement 1). We observed that the two zones are concentric, demonstrating that cortically evoked PAD and motor command of the hindlimb can originate from the same cortical area.

Figure 1. The same cortical area evokes dorsal root potentials (DRPs) and muscular contractions of the hindlimbs.

(A) Schematic experimental design for cortically evoked (Cx-) DRP and electromyographic (EMG) recordings elicited by photostimulation of the contralateral sensorimotor cortex of Thy1-ChR2 mice. DRPs correspond to the presynaptic depolarization of primary afferents propagating antidromically to the suction recording electrode containing a lumbar dorsal root, while EMGs are recorded in the tibialis anterior (TA) muscle. (B) Representative traces of a cortically evoked DRP (top red trace, average of 30 sweeps) and EMG (bottom blue trace, average of three sweeps) after photostimulation (blue window) of the contralateral cortex. (C) Left: heatmaps of the amplitude of the responses of cortically evoked DRPs (top) and EMGs (bottom) in % or the maximum response. Right: overlap of the two maps (red: DRP; blue: EMG) presented as isopotential contour plots (three color grades corresponding to 37, 50, and 63% of maximum value). Coordinates of the cortex are expressed in mm and centered on Bregma. M1: primary motor cortex; M2: secondary motor cortex; S1: primary sensory cortex, according to the Paxinos atlas (Kirkcaldie et al., 2012). (D) Schematic experimental design to identify GAD65-expressing neurons amongst corticospinal neurons targets: AAV2/1-CBA-WGA-CRE was injected in the sensorimotor cortex of TdTomato-flex X GAD65-GFP mice. Analysis of extend of the injection site is presented in Figure 1—figure supplement 1 and of the monosynaptic nature of the transynaptic tracing in Figure 1—figure supplement 2. (E) Photomicrographs (z-projection of confocal images) of a GAD65-GFP mouse lumbar dorsal horn (localization of the view indicated in the inset) after transynaptic labeling from the hindlimb sensorimotor contralateral cortex. A target of the CST (expressing TdTomato after transynaptic transfer of WGA-Cre) also expresses GAD65-GFP. CST: corticospinal tract. Similar experiment in ChAT-EGFP mice is presented in Figure 1—figure supplement 3.

Figure 1.

Figure 1—figure supplement 1. Sensorimotor injection site analyses: cortical layer V fluorescence quantification.

Figure 1—figure supplement 1.

(A) Schematic of experimental design to transynaptically label cortical postsynaptic neurons. (B) Left: drawing of a sagittal slice of the mouse brain illustrating (red square) the localization of the pictures at the right. Right: images of the sensorimotor cortex of a single animal at different lateral positions from the midline. The blue squares show the analyzed area, comprising cortical layer V. The plots show the vertical averages of pixel intensity throughout each blue square. The three slices are localized in the sites indicated by the blue lines in (C). (C) Example of an injection site represented as a heatmap of the pixel intensity across the layer V of a cortex. The map was built using the three plots presented in (B) (illustrated here in the right to show their orientation) as well as additional ones from sequential brain sections on the same animal. (D) Overlap between the heatmap in (C) and the functional area eliciting motor contraction and primary afferent depolarization (PAD; red line). The black line indicates the area with a pixel intensity above 30% .
Figure 1—figure supplement 2. Monosynaptic transynaptic tracing from the cortex.

Figure 1—figure supplement 2.

(A) Schematic of experimental design to transynaptically label cortical postsynaptic neurons. (B) Sagittal image of the injection site in the sensorimotor cortex illustrating TdTomato + neurons (red) and DAPI (blue). The drawing inset shows the area corresponding to the image. (C) Example of stained postsynaptic cortical neurons found in the brain, thalamus, striatum, and red nucleus. The arrow points to TdTomato + neurons. The drawing inset indicates the area corresponding to the image (red square). (D) Absence of TdTomato + neurons in the deep cerebellar nuclei (of a GAD65-GFP mice injected in the cortex as illustrated in A). (E) In the spinal cord, the labeling is restricted to the descending corticospinal tract (CST) (and to its targets) while the ascending tracts are devoided of labeling (CC: central canal). As illustrated in this figure, the targets of the CST are always located in nuclei directly innervated by the cortex, whatever the delay between the injection on me and the observation (see also Figure 4—figure supplement 1). The labeling observed in the spinal cord is exclusively derived from anterograde labeling as transynaptic retrograde transport is not observed in the spinal ascending tracts (nor in the deep cerebellar nuclei). This confirms previous data demonstrating that, when WGA is transgenically encoded (in transgenic mice or by AAVs), the transynaptic labeling is exclusively monosynaptic and anterograde (Libbrecht et al., 2017).
Figure 1—figure supplement 3. Postsynaptic corticospinal neurons do not colocalize with ChAT.

Figure 1—figure supplement 3.

(A) Schematic of experimental design to transynaptically label the targets of the corticospinal tract (CST) in ChAT-GFP transgenic mice. WGA-Cre expressed in the sensorimotor cortex after AAV2/1 infection can be transferred to the targets of the cortex, for example, in the spinal cord; the mice express a Cre-dependent form of TdTomato, so TdTomato will be expressed in the CST targets after anterograde transynaptic transfer of WGA-Cre. In this experiment, the mice are also ChAT-EGFP, thus allowing to test whether (some) CST targets are cholinergic. (B) Images from the contralateral dorsal horn of the spinal cord (z-projection of a stack of confocal images, horizontal section located ~300 μm from the dorsal surface, see inset at the left) showing targets of the CST (TdTomato+, red) and ChAT-GFP+ neurons (green). The arrowheads point at ChAT-EGFP+ neurons located in lamina III and IV, none of which expressed TdTomato. The * points at targets of the CST that do not express ChAT-EGFP.

Spinal targets of the CST

We next investigated the spinal circuit underlying cortically evoked DRPs. DRPs can be segmentally evoked by the activation of a neighboring root, dampening sensory inputs predominantly through GABA-dependent primary-afferent depolarization (Rudomin and Schmidt, 1999). GABAergic terminals presynaptic to proprioceptive fibers arise from GAD65 interneurons (Hughes et al., 2005; Betley et al., 2009) and their activation produces DRPs (Fink et al., 2014). We investigated whether the CST directly targets GAD65-expressing spinal lumbar neurons that could in turn lead to inhibition of primary afferents. In TdTomato-flex mice crossed with GAD65-GFP mice, we injected a monosynaptic anterograde transynaptic virus (AAV2/1-CBA-WGA-Cre) encoding the Cre-recombinase fused to the wheat germ agglutinin (WGA; Libbrecht et al., 2017) in the area inducing hindlimb DRPs and EMG (Figure 1—figure supplement 1). In contrast to its retrograde and anterograde transport when directly injected (LeVay and Voigt, 1990), WGA is reported to provide an exclusively anterograde transneuronal tracing when expressed in transgenic mice (Braz et al., 2002) or virally, including in the form of a WGA-Cre fusion (Gradinaru et al., 2010; Libbrecht et al., 2017), but see Xu and Südhof, 2013. We indeed observed that the fusion with WGA provided transynaptic properties to the Cre recombinase, which was transferred to the cortex’s monosynaptic targets where it triggered the expression of TdTomato (see Materials and methods, Figure 1—figure supplement 2). While a retrograde transynaptic labeling is difficult to exclude in the brain due to the reciprocal connections of the cortex with its targets, the absence of direct spino-cortical projection (and absence of labeling in the ascending tracts, see Figure 1—figure supplement 2E) ensures that spinal Td-Tomato-expressing neurons are direct targets of the CST. We evaluated the proportion of GAD65+ neurons among these neurons (Figure 1D,E): 16.4% ± 3.2% of spinal Td-Tomato-positive neurons were GAD65-GFP+ (69 neurons out of 427, n = 3 mice), suggesting that the cortically evoked DRP may be mediated directly by the activation of a GAD65+ spinal target of the CST. In the spinal dorsal horn, cholinergic neurons are a subpopulation of GABAergic neurons (Todd, 1991; Mesnage et al., 2011), and cholinergic terminals are known to be presynaptic to primary afferents (Ribeiro-da-Silva and Cuello, 1990; Pawlowski et al., 2013) and have recently been shown to modulate primary afferent inputs (Hochman et al., 2010; Shreckengost et al., 2021). By performing a similar experiment in ChAT-EGFP mice, we showed that only 2.2% ± 1.8% of CST targets were ChAT-EGFP positive (1 out of 45 neurons, n = 4 mice), limiting the likelihood of a direct major involvement of a cholinergic mechanism (Figure 1—figure supplement 3).

Segregation of pathways for cortically evoked DRPs and EMGs

Although lumbar DRPs and tibialis-EMGs are elicited by the same area in the mouse sensorimotor cortex, whether they share the same corticofugal pathway and spinal circuits remains unknown. We first interrogated the contribution of indirect cortical-to-spinal pathways to these two functions (Figure 2). Since photostimulation of the sensorimotor cortex in Thy1-ChR2 activates a heterogeneous population of layer V cortical neurons (Arenkiel et al., 2007), we selectively lesioned the direct CST (Figure 2B) using an acute electrolytic lesion at the level of the pyramidal decussation (pyramidotomy) (Figure 2—figure supplement 1A, C). In this study, we have consistently performed a caudal pyramidotomy, sparing CST collaterals targeting brainstem motor nuclei that branch more rostrally (Akintunde and Buxton, 1992; Alstermark and Pettersson, 2014) as well as rubrospinal or reticulospinal tracts that can be activated by corticorubral or corticoreticular neurons (Figure 2B). Cortically evoked DRPs were completely abolished by this pyramidotomy (DRP amplitude before: 3.8 µV ± 0.32 µV; after: 0.30 µV ± 0.12 µV, n = 3 mice, p=0.006), whereas cortically evoked EMGs were hardly affected (S/N ratio 3.3 ± 0.61 before, 2.2 ± 0.1 after, n = 4 mice, p=0.517, all Z-scores above significance after lesion, Figure 2—figure supplement 2B; Figure 2C,D). This demonstrates that indirect cortical-to-spinal pathways (involving supraspinal motor centers) do not encode cortically evoked DRPs but have a major role for cortically evoked motor contraction of the hindlimb. These results show that, in the mouse lumbar cord, the CST mediates cortically evoked DRPs and hence modulates sensory inputs in a presynaptic manner.

Figure 2. The corticospinal tract (CST) is essential for cortically evoked inhibition of primary afferents.

(A) Left: diagram showing direct and indirect cortical-to-spinal paths. In green: paths potentially activated by photostimulation in the cortex of Thy1-ChR2 mice. Right: experimental design. (B) Left: drawing showing the extent of the pyramidal lesion (in gray, pyx) in the frontal plane (distance to Bregma indicated) in two animals whose recordings are respectively illustrated in (C) and (D). Right: diagram showing the indirect cortical-to-spinal paths spared by the pyramidotomy. Systematic histological analysis of the pyramidotomies is presented in Figure 2—figure supplement 1. (C) Left: representative traces of cortically evoked dorsal root potentials (DRPs) recording before (red) and after (gray) CST lesion (average of 30 traces). Right: the pyramidotomy abolishes cortically evoked DRPs (p=0.006, one-way ANOVA, n = 3 mice). Post-hoc test Holm–Sidak, *p<0.05 (D) Left: representative traces of cortically evoked electromyographic (EMG) recording before (blue) and after (gray) pyramidal lesion (average of three traces). Right: there is no significant change in the S/N ratio of the EMG response before and after the pyramidal lesion (93,8, 96,9, and 109,3% prior to pyramidotomy to 77.2% after, p=0.517, one-way ANOVA, n = 4 mice). Gray zone: noise level (see Materials and methods). EMG Z-scores for individual mice before and after pyramidotomy are presented in Figure 2—figure supplement 2.

Figure 2.

Figure 2—figure supplement 1. Histology of the pyramidotomies.

Figure 2—figure supplement 1.

(A) Slices of Thy1-ChR2 mice used for electromyographic (EMG) recordings at the level of the decussation of the pyramids. Extent of the electrolytic lesion in hatched red. (B) Slices of ChR2 retrogradely labeled mice used for EMG recordings at the level of the decussation of the pyramids. Extent of the electrolytic lesion in hatched red. (C) Slices of Thy1-ChR2 mice used for dorsal root potential (DRP) recordings at the level of the decussation of the pyramids. Extent of the electrolytic lesion in hatched red.
Figure 2—figure supplement 2. Z-score of individual electromyographic (EMG) responses assessing the significance of the corresponding S/N ratio.

Figure 2—figure supplement 2.

(A) Matrix of the median Z-score (from five animals) for EMG responses recorded at each stimulated point of the cortex. Data presented in Figure 1C. Coordinates are expressed in mm from Bregma. (B) Z-score of the EMG response for each experimental condition in Thy1-ChR2. Data presented in Figure 2D. (C) Z-score of the EMG response for each experimental condition in mice injected with the ChETA expressing retroAAV. Data presented in Figure 3E. (D) Z-score of the EMG responses for each mouse expressing GFP in corticospinal (CS) neurons. Data presented in Figure 3—figure supplement 1. (E) Z-score of the EMG responses for each mouse expressing ChR2 in the lumbar targets of CS neurons. Data presented in Figure 4G.

Although the contribution of the rubrospinal or reticulospinal tracts to motor command is well acknowledged in both rodents and primates, the CST is also believed to encode motor command (Wang et al., 2017). This could have been underrated by simultaneous stimulation of the indirect cortical-to-spinal pathways in Thy1-ChR2 mice. We thus interrogated the specific contribution of the CST to motor contraction by targeting exclusively these neurons through injection of a ChR2-encoding retrograde virus (AAVrg-CAG-hChR2-H134R-tdTomato) in the lumbar spinal cord (Figure 3A). As expected, the infected CS neurons were located in the area delimited by the previous functional mapping of hindlimb muscle contraction and DRP (Figure 3B,C). Photostimulation of CS neurons (at the cortical level) induced a robust EMG signal (S/N ratio 2.4 ± 0.52, n = 5 mice, Figure 3E, upper), demonstrating that CS neurons evoke motor contraction. In agreement with the pyramidotomy results (Figure 2C), the photostimulation of CS neurons also induced DRPs (mean amplitude = 14.5 ± 4.0µV, n = 5), while photostimulation in control mice expressing GFP in CS neurons induced neither an EMG (all Z-scores below significance) nor a DRP signal (mean amplitude = 0.3 ± 0.0 µV, n = 4, Figure 3—figure supplement 1). Because some CS neurons send collaterals to supraspinal regions involved in motor command (Akintunde and Buxton, 1992), we next addressed whether the motor command traveled through the direct cortical-to-spinal branch of CS neurons or via supraspinal relays. A pyramidotomy, similar to the ones performed above (Figure 2—figure supplement 1C), completely abolished the EMG signal evoked by cortically activating CS neurons (S/N ratio 0.9 ± 0.12; n = 3 mice, p=0.013; all Z-scores below significance after pyramidotomy, Figure 2—figure supplement 2C; Figure 3D,E). This demonstrates that, while supraspinal collaterals of the CST do not trigger motor output, direct spinal branches can drive motor command. However, this contribution does not seem significant when other cortical neurons are co-activated (Figure 2C).

Figure 3. The corticospinal (CS) tract can encode muscular contractions.

(A) Left: diagram illustrating CS neurons and their collaterals expressing ChR2 after retrograde infection in the lumbar cord with a ChR2-retro AAV. Right: experimental design: the photostimulation and electromyographic (EMG) recording session took place at least 3 weeks after the infection. (B) Sagittal image from the sensorimotor cortex (lateral = 0.68 mm), showing retrogradely labeled CS neurons (expressing ChR2-TdTomato). (C) Drawing of a cortex top view, showing the localization of retrogradely labeled CS neurons in the five animals (solid lines). They are within the area functionally determined as inducing dorsal root potentials (DRPs) and EMGs in the hindlimb (dashed lines). (D) Left: drawing showing the extent of the pyramidal lesion in the animal whose recordings are illustrated in (E). Right: diagram showing the supraspinal collaterals of ChR2-expressing CS neurons spared by the pyramidotomy. Systematic histological analysis of the pyramidotomies is presented in Figure 2—figure supplement 1. (E) Left: cortically evoked EMG recordings before (blue) and after (gray) the pyramidal lesion. Right: the pyramidotomy abolishes cortically evoked EMG (p=0.012, one-way ANOVA, n = 5 mice). Post-hoc test Holm–Sidak, *p<0.05. Gray zone: noise level (see Materials and methods). EMG Z-scores for individual mice before and after pyramidotomy are presented in Figure 2—figure supplement 2. The blue window on recording traces indicates photostimulation. SMC: sensorimotor cortex; BSMN: brain stem motor nuclei; C-Th: cervico-thoracic spinal cord; L-SC: lumbo-sacral spinal cord.

Figure 3.

Figure 3—figure supplement 1. Dorsal root potentials (DRPs) are induced by photostimulation of ChR2-expressing corticospinal (CS) neurons (but not by GFP-expressing CS neurons).

Figure 3—figure supplement 1.

(A) Left: diagram illustrating CS neurons and their collaterals expressing ChR2 after retrograde infection in the lumbar cord with a retroAAV. Right: experimental design: the photostimulation and DRP recording session took place at least 3 weeks after the infection. (B) Example of DRP recording obtained after cortical photostimula tion. (C) Analysis of DRP amplitude in the different animal models: THY1-ChR2 (recordings presented in Figures 1 and 2), ChR2-retro (recordings presented in A, B), ChETA expression in the targets of the CS tract (photostimulalation of the spinal cord, Figure 4). (D) Left: experimental design of control experiment consisting of the retrograde infection of CS neurons with a control, GFP-encoding, retrograde AAV. Right: cortical photostimulation induced no DRP or electromyographic signal.

Spinal targets of the CST evoke DRPs but not muscular contraction

Because both DRPs and EMGs of a given limb are encoded by the same sensorimotor cortical area and can be conveyed by the spinal branch of the CST, we next interrogated whether these functions are segregated at the spinal level. CS neurons projecting to the lumbar spinal cord can give off collaterals at the cervico-thoracic level (Kamiyama et al., 2015; Karadimas et al., 2020) and contact cervical propriospinal neurons (Ueno et al., 2012; Ni et al., 2014) that in turn project to lumbar motoneurons to relay motor command (Ni et al., 2014). Indeed, many experiments demonstrated that these neurons are involved in segmental coordination (Miller et al., 1975; Nathan et al., 1996; Reed et al., 2006; Reed et al., 2009). In order to test the contribution of the lumbar branch of CS neurons and rule out antidromic stimulation of rostral collaterals, we combined the transynaptic tool used in Figure 1 with an intersectional approach at the lumbar level. We first identified the lumbar segments containing CST targets by using cortical injection of AAV2/1-CBA-WGA-Cre in TdTomato-flex mice (Figure 4A): CST targets were concentrated in spinal lumbar segments L2–L3, rostral to the large pools of motoneurons. They were largely located in the dorsal horn (85.4% were dorsal to the central canal), in particular in the contralateral deep dorsal horn (laminae IV to VII) (Figure 4B, Figure 4—figure supplement 1). Consistently with the monosynaptic nature of the transynaptic labeling, their number was not correlated with the delay between the infection and the histological analysis (Figure 4—figure supplement 1). CST targets were mostly located in the medial zone, lateral to the dorsal funiculus, where CST fibers penetrate the gray matter. We thus combined the cortical injection of AAV2/1-CBA-WGA-Cre with the spinal (L4) injection of an AAV (AAV9-Ef1a-DIO-ChETA-EYFP) encoding for a Cre-dependent form of ChETA (a ChR2 variant Gunaydin et al., 2010; Figure 4C). ChETA-EYFP expression was therefore restricted to the lumbar targets of the CST (Figure 4C and D). Our transectional approach induced ChETA-EYFP expression even in the deepest targets of the CST in the ventral horn (Figure 4—figure supplement 2). There was no ChETA-EYFP labeling in the dorsal funiculus, demonstrating that the intraspinal AAV injection did not lead to retrograde infection of CS neurons (Figure 4—figure supplement 2). Intraspinal injections did not affect the general behavior of mice (e.g., no visible motor deficits nor pain-like behaviors) nor induced long-lasting weight loss. As surface photostimulation of the spinal cord in Thy1-ChR2 animals produced an EMG signal and directly activated neurons even in the deep dorsal horn (Figure 4—figure supplement 3), we performed an identical photostimulation in the animals expressing ChETA in the targets of the CST. This stimulation induced a spinal LFP observable as deep as 1150 µm from the surface (Figure 4—figure supplement 3). Although the number of ChETA-expressing neurons was highly variable (due to the variability inherent to the WGA-Cre transynaptic tool, see Figure 4—figure supplement 1, and to the intersectional strategy), spinal photostimulation induced DRPs in the ipsilateral lumbar root of eight out of nine mice (2.93 ± 0.48 µV, n = 8 mice, Figure 4E–G), demonstrating that DRPs can be directly controlled by the CST projecting to the lumbar cord through local interneurons. Interestingly, the amplitude of the DRPs was not related to the number of ChETA-expressing spinal neurons, suggesting that probably only a fraction of those are involved in primary afferent control. However, photostimulation of these same lumbar targets of the CST (same animals) failed to elicit hindlimb movements or muscle contraction as attested by EMG recordings (S/N ratio 1.0 ± 0.02; n = 7 mice, Z-scores below significance, Figure 2—figure supplement 2D; Figure 4E–G). Together with the results presented in Figure 3, we can conclude that CS neurons are able to evoke motor contraction by activation of their spinal targets, but that these targets are not located in the lumbar cord but rather at another spinal level. Cortically evoked DRPs and EMGs thus follow a similar corticofugal pathway that segregates at the spinal level.

Figure 4. Lumbar corticospinal postsynaptic neurons encode dorsal root potentials (DRPs) but no movements.

(A) Experimental design: the targets of the corticospinal tract (CST) are labeled through a transynaptic approach consisting of AAV2/1-CBA-WGA-CRE injection in the hindlimb sensorimotor cortex of TdTomato-flex mice. (B) Localization of the spinal targets of the CST: heatmap showing the distribution of the neurons in the lumbar cord (6 mm long) projected into the transverse plane (average of nine mice; left) or horizontal plane (average of six mice; right) plane. (C) Left: diagram illustrating that only lumbar direct targets of the CST express CHETA in the following experiment. Right: experimental design: TdTomato-flex mice received an injection of AAV2/1-CBA-WGA-CRE in the hindlimb sensorimotor cortex and an injection of AAV9-Efl-flex-CHETA-eYFP in the L4 spinal segment. (D) Photomicrographs (z projection of confocal images) from the dorsal horn of the spinal cord (laminae V/VI); the arrows point at two targets of the CST expressing TdTomato+ and CHETA-eYFP. (E) Experimental design illustrating spinal photostimulation of the lumbar targets of the CST. (F) Representative traces of DRP (red trace, average of 60 traces) and electromyographic (EMG; blue trace, average of three traces) recordings from the same animal after photostimulation of the lumbar targets of the CST (blue window). (G) Photostimulation of the lumbar targets of the CST induces DRPs (left) but no EMG signal (right). Gray zone: noise level (see Materials and methods). EMG Z-scores for individual mice are presented in Figure 2—figure supplement 2. SMC: sensorimotor cortex; BSMN: brain stem motor nuclei; C-Th: cervico-thoracic spinal cord; L-SC: lumbo-sacral spinal cord.

Figure 4.

Figure 4—figure supplement 1. Strategy to label the spinal targets of corticospinal (CS) neurons.

Figure 4—figure supplement 1.

(A) Experimental design: the targets of the corticospinal tract (CST) are labeled through a transynaptic approach consisting of AAV2/1-CBA-WGA-CRE injection in the hindlimb sensorimotor cortex of TdTomato-flex mice. (B) Dorsoventral distribution of TdTomato neurons in the spinal cord after transynaptic labeling. N = 570 neurons from nine animals. The 0 coordinate corresponds to the center of the central canal. 85% of neurons are located dorsally to the central canal. (C) Total number of TdTomato neurons in the lumbar spinal cord (systematic counting) in the different animals considered for building Figure 4A, including indication of survival delay after cortical injection, and infected area (in mm2) in the cortex after histological analysis. These parameters do not correlate with the number of TdTomato neurons as demonstrated by the low Pearson coefficient.
Figure 4—figure supplement 2. Strategy to stimulate exclusively the spinal targets of corticospinal (CS) neurons.

Figure 4—figure supplement 2.

(A) Experimental design: the targets of the corticospinal tract (CST) are labeled through a transynaptic approach consisting of AAV2/1-CBA-WGA-CRE injection in the hindlimb sensorimotor cortex of TdTomato-flex mice. (B) Number of ChETA-expressing targets of the CST and size of dorsal root potential (DRP) signal recorded in the corresponding mice (no correlation). These numbers are underestimated as the histological analysis was performed after a long recording session, with only post-fixation (no intracardiac perfusion of fixative). (C) After the transectional approach presented in (A), the spinal postsynaptic targets of CS neurons express ChETA-eYFP (two *). Importantly, the dorsal funiculus, and in particular the CST, is not labeled by this approach. CC: central canal. (D) The spinal CS postsynaptic neurons (that received the transynaptic WGA-Cre) infected by the intraspinal AAV injection (encoding ChETA-flex) can be located in the ventral horn, as in this example of a neuron located at 180 μV ventral to the CC. Neurons up to –230 μV from the CC were infected, that is, a depth where 97% of the spinal targets of the CS neurons are found (Figure 4—figure supplement 1B).
Figure 4—figure supplement 3. Efficacy of the surface spinal photostimulation.

Figure 4—figure supplement 3.

(A) Experimental design (left) for LFP recordings (right): the lumbar targets of the corticospinal tract (CST) express ChETA after intersectional viral strategy, and LFP recording are obtained at different depth after spinal surface photostimulation. (B) Experimental design (left) for spinal single unit (right top) and electromyographic (EMG; right bottom) recordings in Thy1-ChR2 mice, after spinal surface photostimulation (isoflurane anesthesia). (C, left) Depth of neurons recorded by juxtacellular single-unit recordings, directly responding to the surface illumination (1 ms or 0.5 ms pulse). Right: Criteria to establish that these neurons were directly activated: the delay of the response was lower than 5 ms, the standard deviation of this delay was lower than 0.20 ms, and (not shown) there was no failure (or less than 0.5% of failure) when stimulated at 4 Hz. (D) Example of a spinal neuron located at 500 μm from the cord surface. (D1) Superimposition of electrophysiological traces of the photostimulated action potential. (D2) Raster plot of the 1000 episodes illustrating the stability of the response and of its delay. (D3) Peristimulus histogram presenting the distribution of the spike delay of this neuron in the 1000 episodes presented in (D2). Note: In contrast to the spinal photostimulation presented here, cortical photostimulation of THY1 animals under isoflurane anesthesia did not lead to an EMG signal; this was only obtained under ketamine/xylazine anesthesia.

Discussion

In this study, in order to gain knowledge on the contribution of the CST to the cortical control of movements, we have evaluated its ability to convey the muscle contraction command or to modulate sensory inputs. We found that the modulation of sensory inputs from the hindlimb (DRP in lumbar sensory roots) is exclusively carried out by the CST via a population of lumbar interneurons located in the deep dorsal horn. In contrast, the cortex induces muscle contraction of the hindlimb (TA) through a relay in the upper spinal cord or supraspinal motor centers. The two mechanisms are therefore segregated, and the main role of the lumbar CST appears to be the modulation of sensory inputs.

Reliability of circuit investigations

We have identified the cortical area involved in these two mechanisms through functional means: cortically evoked DRPs and EMGs. For ethical and technical reasons, most of these recordings were performed on different animals and conditions. First, EMG recordings after cortical stimulation required a light anesthesia level (Tennant et al., 2011), only possible using ketamine/xylazine. Such a light anesthesia is not ethically compatible with the large and invasive surgery required for recording cortically evoked DRPs. DRPs were thus recorded under isoflurane anesthesia under which no cortically evoked EMG could be recorded. We therefore acknowledged that the cortical zone responsible for DRPs, smaller than the one responsible for EMGs, might have been underestimated by the deeper anesthesia. We thus did not attempt to quantitatively compare EMG vs. DRP map extents, but concluded that they partially overlap allowing us to stimulate at the center of two maps. Although the different experimental approaches might also have differentially affected the recruitment threshold for EMG vs. DRP, we do not compare these thresholds in between stimulation/recording configurations. Rather, we analyze the amplitude of a given signal (EMG or DRP) with a given experimental approach in different animal models (THY1, ChR2-retro, pyramidotomy, transynaptically labeled CS targets).

Circuit investigation also relies on the efficiency of pyramidotomies that affect the spinal branch of CS tract while sparing CST collaterals targeting brainstem motor nuclei that are branching more rostrally (Akintunde and Buxton, 1992). We performed similar pyramidotomies in two different group of mice, either Thy1-ChR2 or ChR2-retro group (Figures 1 and 2, Figure 1—figure supplement 3). In the latter group, in which the CST expressed ChR2, we observed a complete loss of cortically evoked EMGs after pyramidotomy. These results confirm that the pyramidotomy was efficient to block CST-induced EMG. Therefore, the remaining EMG signal after pyramidotomy in Thy1-ChR2 mice (Figure 2D) is likely the result of a non-CST pathway.

Finally, an intriguing result we report is the absence of EMG signal after photostimulating the targets of the CST at the lumbar level (Figure 2F,G). The reliability of this result relies on multiple technical controls. First, the lack of muscle contraction cannot be attributed to damages of spinal microcircuits due to intraspinal AAV injection. None of the mice presented obvious alteration in the behavior nor a long-lasting weight loss. In addition, similar intraspinal injections were performed for the ChR2-retrograde group, and in these animals, EMG was systematically observed after cortical stimulation, demonstrating the integrity of required spinal circuits. Second, the intraspinal AAV injection efficiently infected neurons in the dorsal horn, and even in the dorsal part of the ventral horn, that is, in an area comprising 97% of the lumbar targets of the CST. Third, the optic fiber positioned at the surface of the cord evoked an intraspinal LFP as deep as 1150 µm in the double-infected mice, but also evoked EGMs in Thy1-ChR2 mice, as previously reported (Caggiano et al., 2016). We also demonstrate direct photoactivation of neurons located up to 700 µm from the surface with the short 1 ms stimulus used for EMG recordings. As 73% of the targets of the CST are located within 700 µm from the surface, this demonstrates that the large majority of this population can be directly activated by this procedure. Finally, using a similar surface photostimulation with powers equal to or lower than in the present study, Caggiano and colleagues were able to induce motor contraction in ChAT-ChR2 animals and interpreted this as direct activation of motoneurons located in the deep ventral horn (Caggiano et al., 2016). We thus consider very likely that the surface illumination we used could reach most of the lumbar CS-postsynaptic neurons, and that the absence of EMG in this situation is biologically relevant.

Anatomical definition of the hindlimb sensorimotor cortex

In our study, the cortical area inducing hindlimb contraction is similar to that previously reported in mice (Li and Waters, 1991; Ayling et al., 2009). This cortical area (centered around 1 mm caudal and 1.5 mm lateral to Bregma) spans over the motor cortex and also partly over the primary somatosensory cortex according to some atlases and studies (Kirkcaldie et al., 2012; Liu et al., 2018; Karadimas et al., 2020). We thus use the conservative term ‘hindlimb sensorimotor cortex.’ Interestingly, we found that stimulation of this area also induced PAD at the lumbar level. This overlap might be a specificity of the ‘hindlimb area’ of the cortex (HLA) and may not generalize to the ‘forelimb areas’ (RFA and CFA). Indeed, the CS neurons from the forelimb areas are spatially segregated (forming clusters projecting either to the dorsal or to the intermediate-ventral cervical cord), while those from the HLA are largely intermingled (Olivares-Moreno et al., 2017). In addition, our results demonstrate that the same cortical area is labeled after retrograde tracing from the lumbar cord (Figure 3C) excluding the existence of a different pool of cortico-lumbar neurons.

Pathways conveying the cortical motor command

Our study concludes that a large fraction of the cortical motor command to the hindlimb is conveyed through non-CST pathways. These could involve the reticular formation or the red nucleus that receive cortical innervation, project directly or indirectly onto spinal motoneurons, and control different parameters of movements (Lavoie and Drew, 2002; Zelenin et al., 2010; Esposito et al., 2014). Our recordings also suggest that the cortex conveys a motor command to the hindlimb through the CST projecting to a non-lumbar spinal segment as direct stimulation of the CS lumbar targets failed to produce muscle contraction. We have discussed above possible technical caveats and concluded that our approach most likely induced activation of the vast majority of the lumbar CS targets labeled by the transynaptic tracing. We cannot rule out that some of these lumbar targets contribute to convey the cortical motor command to the hindlimb but were not labeled in large enough quantities through our transynaptic approach. The efficiency of WGA-Cre transynaptic tracing might be target specific (Libbrecht et al., 2017), and the number of labeled neurons in the spinal cord suggests that not all targets were labeled. However, this would still implicate that these neurons are distinct from the ones involved in the generation of a DRP, and that these two functions use segregated lumbar pathways. While this hypothesis cannot be excluded, the recent report by Karadimas and collaborators (Karadimas et al., 2020) strongly supports the hypothesis that the motor cortical command conveyed by the CST relays in rostral spinal segments. Indeed, they demonstrate that the cortical motor command to the hindlimb can be mediated by CS neurons projecting to the cervical cord, and then relayed by cervical-to-lumbar propriospinal neurons (Karadimas et al., 2020). Although we selectively study CS projecting at least to the lumbar cord (through lumbar retrograde infection), many CS neurons have collaterals at different spinal segments (Kamiyama et al., 2015). Therefore, their photostimulation at the sensorimotor cortex level would activate cervical pathways in addition to lumbar ones.

Pathways conveying the cortical sensory control

Beyond inducing hindlimb muscle contraction, we have characterized the ability of the cortex to induce DRPs, which is a measure of PAD (Wall and Lidierth, 1997). We demonstrate that it is exclusively relayed through the CST and provide some information on spinal interneurons potentially mediating this effect. The most studied mechanism of segmental PAD involves a presynaptic GABAergic last order neuron (mostly expressing GAD65; Betley et al., 2009), located in laminae V–VI, under the control of a first-order excitatory interneuron that receives peripheral and descending (including cortical) inputs (Eccles et al., 1962; Rudomin and Schmidt, 1999; Betley et al., 2009). The targets of the CST identified by our transynaptic labeling are indeed predominantly located in laminae IV–VI and include a small population of GAD65-expressing neurons. They represent a potential substrate for cortically evoked DRP that would thus not require a spinal first-order excitatory interneuron.

In addition to the sensorimotor cortex, stimulation of other supraspinal nuclei is known to induce PAD. This is the case of the red nucleus and the reticular formation (Jiménez et al., 1987; Sirois et al., 2013), both of which also receive inputs from the sensorimotor cortex. The corticorubral and corticoreticular tracts were preserved by the pyramidotomy, yet the cortically induced PAD was completely lost. This strongly suggests that the ability of the red nucleus and reticular formation to induce PAD does not rely on a sensorimotor cortical command.

The control of incoming sensory information through PAD plays an important role in the generation of coordinated movements and processing of sensory information, including touch and nociceptive information {Liu et al., 2018, #2735; McComas, 2016, #2869}. GAD65-expressing neurons are essential for smoothed forelimb movements (Fink et al., 2014), but whether they require peripheral or cortical information to do so is yet unknown. Similarly, recordings in primates demonstrated that sensory information is inhibited in a modality- and phase-dependent manner during forelimb voluntary movements (Seki et al., 2003; Seki et al., 2009; Seki and Fetz, 2012), suggesting that this inhibition is somehow related to the motor command, but the exact underlying mechanism remains to be elucidated. Importantly, the change in afferent fiber excitability (Wall, 1958) accompanying the sensory presynaptic inhibition in primates was even smaller (<2 µV; Seki et al., 2003) than the DRPs we report as a consequence of cortical stimulation, suggesting that cortically evoked DRPs might be sufficient to significantly impact the transmission of sensory information. Here, we demonstrate that CS neurons from the same sensorimotor cortex area (but not necessarily a single population) are able to relay both motor command and sensory modulation to hindlimb, but through segregated pathways. Future studies on awake animals and/or focusing on specific sensory modalities are needed to further elucidate the timing and specificity of the sensory control we have described.

Significance of the results

In contrast with the present results on hindlimbs, specific stimulation of the CST at the cervical level induces forelimb contraction (Gu et al., 2017), suggesting a direct relay of the cortical motor command at this level (although antidromic activation of a collateral pathway cannot be excluded). Relative to the forelimb, mice do not display an extensive repertoire of skilled hindlimb movements and a difference in the level of cortical control is expected. The functional segregation we report here suggests that, while the sensory control is selectively targeted at the lumbar level, the hindlimb motor command is relayed in the upper cord. The fact that the upper cord (possibly the cervical cord according to Karadimas et al., 2020), receiving the motor command from the forelimb sensorimotor cortex, also receives the information of hindlimb motor command offers a substrate for the coordinated control of fore- and hindlimb, which is essential for most, if not all, skilled movements.

The organization of the CST greatly differs between species. From an anatomical point of view, this tract travels mainly in the dorsal funiculus in rodents, while it is located in the dorsolateral funiculus in primates, and its projection area within the spinal cord accordingly differs (Lemon, 2008). The evolution of the CST has been linked to the development of the most dexterous movements that can be performed by primates, with the appearance of direct cortico-motoneuronal (CMN) contacts that are absent in rodents. From an evolutionary perspective, it is interesting to note that the lumbar branch of the CST, which will later acquire a direct motor command role (including with direct CMN contacts in primates Porter, 1985), instead has predominant projections in the rodent dorsal horn for preferential modulation of afferent pathways. Importantly, primate CS neurons do not only form CMN contacts but also abundantly project onto spinal interneurons, and the sensory control function of the CST we report has also been proposed in primates (Lemon, 2019). While extrapolation of our results to primates is difficult in view of the large species differences, mice are the main and foremost animal model, including for pathologies affecting the CST such as spinal cord injury or amyotrophic lateral sclerosis. A better understanding of the differential roles of the mouse CST in the cervical and lumbar cord is thus an essential endeavor to properly interpret data from these models.

Conclusion

Altogether, our results support a segregation of pathways involved in cortically evoked sensory modulation vs. motor control. The direct CST is able to induce motor contraction, independently of its supraspinal collaterals, through spinal targets possibly located at the cervico-thoracic level. However, we show that motor command is largely mediated by non-CST pathways, most likely cortico-rubral, or -reticular ones. On the other hand, the major and essential role of the lumbar rodent CST appears to be hindlimb sensory modulation at the primary afferent level through a population of lumbar target neurons whose activation is sufficient to produce DRPs. This forces reinterpretation of previous studies aiming at promoting CST function in a therapeutic perspective, in mouse injury or neurodegenerative models. While most of these studies have exclusively considered motor command aspects, our results provide a new perspective to analyze these efforts by considering the primary role of rodent lumbar CST, that is, sensory modulation.

Materials and methods

Animal models

This study was carried out in strict accordance with the national and international laws for laboratory animal welfare and experimentation and was approved in advance by the Ethics Committee of Strasbourg (CREMEAS; CEEA35; agreement number/reference protocol: APAFIS#12982-2017122217349941v3). The following mice strains were used (adult males and females): thy1-ChR2 (Cg-Tg(Thy1-COP4/EYFP)18Gfng/J, Jackson Laboratory stock no: 007612), TdTomato-Flex (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Laboratory stock no: 007914, Ai14), ChAT-EGFP (von Engelhardt et al., 2007), and GAD65-GFP that stands for gad2-GFP (López-Bendito et al., 2004). All lines except gad2-GFP were back-crossed (at least 10 generations) on a CD1 background. gad2-GFP were on a C57Bl/6J background. The mice were housed in the Chronobiotron facility (UMS3415, CNRS, University of Strasbourg) in accordance with the European convention 2010/63/EU on the protection of animals used for scientific purposes.

Electrophysiological recordings

EMG recording

Ketamine/xylazine anesthesia was chosen to minimally affect muscular tone. After the initial anesthesia (150 mg/kg of ketamine, Imalgene 1000, and 10 mg/kg of xylazine, Rompun 20%), complementary doses of ketamine were given when the animal was showing signs of awakening (additional doses bridged by short exposure to isoflurane 1–1.5%; Tennant et al., 2011). Concentric EMG needle electrodes (Myoline Xp) were inserted in the TA muscle of the animals and a ground electrode was inserted in the tail. Responses were collected with a recording amplifier (IR183A, Cygnus Technology) in series with a second amplifier (Brownlee Precision, model440). The traces were filtered with a bandwidth of 0.1 Hz to 10 kHz, recorded with Spike2 software (version 8.00, CED, Cambridge, UK), and analyzed off line with Clampfit (pCLAMP, version 10.7) and home-made Python routines (WinPython 2.7.10, Python Software Foundation). EMG responses were recorded after cortical or spinal photostimulation (see below), and the response to three consecutive stimulations was averaged for analysis. The area under the curve of the rectified average trace was calculated in a window of 30 ms, either 40 ms before (baseline noise, N) or 10 ms after (signal, S) the onset of the stimulation. The EMG response was presented as the S/N ratio. In CST lesion experiments, all S/N ratios were expressed as a % of the control S/N (average of three responses before the lesion). The noise level (S/N of 1) was similarly expressed as a % of the above-defined control S/N.

DRP recording

Under isofluorane (1.5–2%) anesthesia, the mice spinal cord was fixed with the spinal cord unit of a stereotaxic frame (Narishige Instruments). A laminectomy was performed to expose the surface of lumbar L3–L6 spinal cord segments. A dorsal root (L4–L6) was dissected, cut before the DRG, and a rootlet was suctioned with a glass pipet (sometime two rootlets were suctioned together). An agar pool was created on the exposed spinal cord and filled with NaCl 0.9%. The amplifiers/filters/software used were the same as for the EMG recordings. DRPs in response to 60 successive cortical or spinal photostimulations (see below) were averaged for analysis. The amplitude of the response from the onset to the peak was measured using Clampfit 10.0. The amplitude of the noise was similarly measured on a window preceding photostimulation. For spinal photostimulation, DRPs and EMGs recordings of three series of photostimulation were averaged.

For electrophysiological recordings, we excluded animals that had a constant decreasing signals (run-down) in otherwise stable conditions. For DRP recordings that required a delicate surgery, animals were excluded from the study if the surgical preparation/recovery was compromised or if scaring was abnormal after intraspinal AAV injection.

Single-unit extracellular recording

The animal was prepared and anesthetized as for DRP recordings (except the dorsal root dissection). Single-unit extracellular recordings were made with a glass electrode (Harvard Apparatus, Holliston, MA, USA) filled with 0.5 M NaCl and 0.06 M HEPES, pH adjusted at 7.3 (resistance ~19 MΩ). A motorized micromanipulator (Narishige, Tokyo, Japan) was used to gradually insert the electrode with 4 µm steps. The amplifiers and software used were the same as for the DRP and EMG recordings, but the signal was at 0.1–3 kHz. The signal was analyzed with Python routines for spike detection (amplitude threshold of 0.30 mV) and delay measure (from onset of photostimulation to spike peak).

Cortical photostimulation

Photostimulation of the cortex was performed on anesthetized animals while recording EMG or DRP. For the cortex, a craniotomy of approximately 9 mm2 was performed under stereomicroscope on the contralateral side from the recording site. Muscle contraction, measured by EMG, was evoked by photostimulation with a 250 µm probe (473 nm laser source PSU-III-FDA, 56 mW/cm²) using the following protocol: trains of six pulses, 1 ms duration, 2 ms interval; 15 s minimum between each train (Li and Waters, 1991; Carmel and Martin, 2014). Cortically evoked DRPs typically require trains of electrical stimulations (Andersen et al., 1962; Andersen et al., 1964; Eguibar et al., 1994; Wall and Lidierth, 1997), a protocol that we adapted for photostimulation using a 105 µm probe (460 nm LED source, Prizmatix, 3.36 mW/cm²): trains of five pulses of 8 ms, 1 ms interval and 2 s between each train. In both cases, the probe was moved at the surface of the cortex along a 500-µm-spaced grid. Isopotential contour plots were created, using for EMGs the S/N and for DRPs the peak amplitude (both normalized to the largest values in each animal) at each photostimulation point in the cortex, using a linear interpolation on a set of X, Y, Z triples of a matrix. For EMG, only S/N ratio above significance (Z-score >3, Figure 2—figure supplement 2A) was considered for the analysis. Then the isopotential contours were superimposed on the metric planes of the top view of the cortex (Kirkcaldie et al., 2012).

Spinal cord photostimulation

Photostimulation of the spinal cord was performed on anesthetized mice after laminectomy (described above for DRPs recordings) using a 1.1 mm probe (LED source, 42 mW/cm²) first placed on the surface of the lumbar spinal cord (on top of the injection site). The stimulation trains were those used for cortical stimulation. If no signal was observed when stimulating on top of the injection site, the optical fiber was then swept rostrally and caudally on top the whole lumbar segment in order to try and obtain a signal. EMG and DRP recordings were sequentially performed and required to alternate between different types of anesthesia: isoflurane (craniotomy and laminectomy surgery), ketamine/xylazine (EMG recording), and isoflurane 1.5–2% (DRPs recordings). Only the mice filling the two following criteria were kept for analysis: (1) TdTomato+/EYFP+ neurons were found in the stimulated area and (2) the response was not precisely associated to the onset or offset of optical pulses (which would be expected for a photoelectric artifact).

For spinal single-unit extracellular recordings, the photostimulation protocol consisted in either a single 1 ms pulse or in the same train used to test EMG responses (six pulses, 1 ms duration, 2 ms interval).

Pyramidotomy

Electrolytic lesion of the CST was performed in a subset of Thy1-ChR2 and ChR2 retrogradely labeled mice, with 200 ms pulses, 30 mA using a constant current stimulator (Digitimer Ltd) through a silver bipolar electrode inserted in the pyramidal decussation (3.5–4 mm caudal to Bregma, 6 mm deep). The brain was removed for histological analysis at the end of the experiment.

Cortical and spinal injections

Brain injections (1.5–2 mm lateral, 0.5–1 mm caudal to Bregma, 0.5 mm deep) were performed as previously described (Cetin et al., 2006) under isoflurane anesthesia (2–3%). Briefly, 90–270 nL of virus was injected by manual pressure using a 5 mL syringe coupled to a calibrated glass capillary under visual control. Spinal injections were performed using a similar manual pressure protocol. The pipette was inserted in the exposed space between two vertebrae (T13-L1, corresponding to spinal L4), as previously described (Tappe et al., 2006). 0.45 µL of virus was injected 300 µm lateral to the midline and 300–400 µm deep. In both cases, Manitol 12.5% was injected i.p. (0.2–0.5 mL) after the surgery to enhance vector spread and improve transduction (Tjølsen et al., 1992). After spinal injection, transfected neurons were found on multiple segments centered in L4 (depending on the animals, up to the whole lumbar enlargement).

The animals were kept 2 weeks for the retroAAV injections and a minimum 5 weeks for dual injections before in vivo DRPs and/or EMGs recordings or histological analysis. Animals for which post-hoc histological analysis demonstrated inappropriate injections coordinates were excluded from the analysis.

Viruses

AAV2/1-CBA-WGA-CRE-WPRE was purchased at the molecular tools platform at the Centre de recherche CERVO (Québec, Canada) and was used at a titer of 8 × 1012 vg/mL. AAV-Ef1a-DIO ChETA-EYFP was a gift from Karl Deisseroth (Gunaydin et al., 2010; Addgene viral prep # 26968-AAV9; http://n2t.net/addgene:26968; RRID:Addgene_26968) and was used at a titer of 1 × 10¹³ vg/mL. AAV-CAG-hChR2-H134R-tdTomato was a gift from Karel Svoboda (Mao et al., 2011; Addgene viral prep # 28017-AAVrg; http://n2t.net/addgene:28017; RRID:Addgene_28017) and was used at a titer of 7 × 1012 vg/mL. pAAV-hSyn-EGFP was a gift from Bryan Roth (Addgene viral prep # 50465-AAVrg; http://n2t.net/addgene:50465; RRID:Addgene_50465) and was used at a titer of 1 × 10¹³ vg/mL.

Histology

Mice were transcardially perfused with PB followed by 4% paraformaldehyde (PFA) in PB 0.1 M, or, if histological analysis followed electrophysiological recordings, the brain and spinal cord were post-fixed overnight in PFA 4% in PB 0.1 M. Serial 50 µm brain (coronal or sagittal) and spinal (transverse or sagittal) sections were performed on a vibratome (Leica VT1000 S) and mounted using a DAPI staining mounting medium (Vectashield, Vector Laboratories).

Image analysis

The extent of the cortical injection site was estimated by measuring the spread of reporter protein fluorescence (TdTomato) in the layer V of the cortex. The intensity of fluorescence was analyzed as previously described (Lorenzo et al., 2008; Mesnage et al., 2011) on evenly spaced (250 µm apart) transverse brain sections imaged with a microscope (Axio Imager 2, Zeiss). Briefly, the ‘Plot Profile’ function of ImageJ software (W. Rasband, National Institutes of Health) was used to measure the intensity of fluorescence along the horizontal axis of a 6000 × 250 µm rectangle containing the layer V of the cortex (Figure 1—figure supplement 1). These values were normalized to the highest intensity for each animal and used to plot a density map of each injection site. The contour of the injection area was defined as 30% of the maximal intensity, corresponding to the approximate intensity of individual neurons.

Spatial distribution of CS postsynaptic neurons

Spinal lumbar sections (50 µm) were mounted serially and imaged using a Zeiss epifluorescence microscope or a Leica SP5 II confocal microscope. Images were aligned manually with Photoshop using the central canal and the dorsal funiculus as landmarks. Each labeled neuron was assigned coordinates corresponding to its distance to the center of the central canal (X and Y) and the index of the slice containing it (Z). This was used to calculate the number of labeled neurons every 100 µm × 100 µm bins (X × Y) (or 200 µm × 200 µm X × Z) in order to build density plots. The neuronal distribution was plotted using the ‘spline 16’ interpolation method of the matplotlib library in a homemade Python script.

Quantification and statistical analysis

No prior sample size calculation was performed. The mean and standard deviation of the noise were measured from the three noise values obtained from each EMG recording and used to calculate the Z-scores. The Z-score of each EMG response to the stimulation was calculated using the following equation:

Zscore=Signal- mean Noisestandard deviation of Noise

A Z-score of 1.96 or 3 corresponding to a significance level of 0.05 or 0.001 was chosen to discriminate significant responses from nonsignificant ones. The conservative choice of threshold was chosen in agreement with visual inspection of the traces to ensure that any EMG responses was not simply due to noise. For each tests, the normality and variance equality were verified by SigmaPlot (version 13, 2014 Systat Software, Inc) using Shapiro–Wilk and Brown–Forsythe’s methods, before we applied the parametric tests. All the data were analyzed using a one-way ANOVA and a post-hoc test was performed only if an effect showed statistical significance. The p-values for multiple comparison were measured using Holm–Sidak’s method. Error bars in all figures represent mean ± SEM, *p<0.05, NS indicates no statistical significance (p≥0.05). The power of the tests was systematically attested to be above the desired power of 0.80.

Acknowledgements

We gratefully acknowledge the support from the CNRS and Strasbourg University; University of Strasbourg Institute for Advanced Study (USIAS) and ANR-13-JSV4-0003-01 grants to MCE; ANR-15-CE37-0001-01 CeMod, ANR-19-CE37-0007-03 MultiMod, and ANR-19-CE16-0019-01 NetOnTime to PI. YML was supported by a postdoctoral fellowships from the Fondation pour la Recherche Médicale (SPF20160936264) and CB was funded by a fellowship from the Ministère de la Recherche and by the ARSLA (Association pour la Recherche sur la SLA). CB was a student of the EURIDOL graduate School of Pain (University of Strasbourg and ANR-17-EURE-0022). We also thank the following for providing/generating specific mouse lines: Gábor Szabó for GAD65-GFP, Jakob von Engelhardt for ChAT- EGFP. We thank Dr. Sophie Reibel-Foisset and the staff of the animal facility (Chronobiotron, UMS 3415 CNRS and Strasbourg University) for technical assistance. We also thank Yves De Koninck, Frédéric Doussau, and Didier Desaintjan for critical reading of the manuscript.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Matilde Cordero-Erausquin, Email: cordero@unistra.fr.

Muriel Thoby-Brisson, CNRS Université de Bordeaux, France.

Ronald L Calabrese, Emory University, United States.

Funding Information

This paper was supported by the following grants:

  • University of Strasbourg Institute for Advance Sciences USIAS fellow to Matilde Cordero-Erausquin.

  • Agence Nationale de la Recherche ANR- 13-JSV4-0003-01 to Matilde Cordero-Erausquin.

  • Agence Nationale de la Recherche ANR - 19-CE37-0007-03 MultiMod to Philippe Isope.

  • Agence Nationale de la Recherche ANR - 19-CE16-0019-01 NetOnTime to Philippe Isope.

  • Fondation pour la Recherche Médicale Postdoctoral fellowship to Yunuen Moreno-Lopez.

  • Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation Graduate Student fellowship to Charlotte Bichara.

  • Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres Maladies du Motoneurone Graduate Student fellowship to Charlotte Bichara.

  • EURIDOL graduate school ANR-17-EURE-0022 to Charlotte Bichara.

Additional information

Competing interests

None.

none.

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review and editing.

Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – review and editing.

Formal analysis, Investigation, Visualization.

Conceptualization, Writing – review and editing, Funding acquisition, Methodology.

Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Validation, Visualization.

Ethics

This study was carried out in strict accordance with the national and international laws for laboratory animal welfare and experimentation and was approved in advance by the Ethics Committee of Strasbourg (CREMEAS; CEEA35; agreement number/reference protocol: APAFIS# 12982 - 2017122217349941 v3).

Additional files

Transparent reporting form

Data availability

All raw data for each figure have been made available on Zenodo.

The following dataset was generated:

Moreno-Lopez Y, Bichara C, Delbecq G, Isope P, Cordero-Erausquin M. 2021. The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord (Raw data) Zenodo.

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Decision letter

Editor: Muriel Thoby-Brisson1
Reviewed by: David Bennett, Klas Kullander, Shawn Hochman2

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This publication is of interest to scientist within the field of motor control and central modulation of sensory inputs, with original data likely to have a major impact on our understanding of the function of the corticospinal tract. By combining optogenetic stimulation with in vivo electrophysiology, it demonstrates that the corticospinal tracts' main direct action on the lumbar spinal cord is to modulate sensory transmission via primary afferent depolarization. Derived insights will help guide future studies on the organization of corticospinal modulation of sensory inputs.

Decision letter after peer review:

Thank you for submitting your article "Lumbar corticospinal tract in rodents modulates sensory inputs but does not convey motor command" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: David Bennett (Reviewer #1); Klas Kullander (Reviewer #2); Shawn Hochman (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Summary:

The study by Cordero-Erausquin investigates pathways for actions of the corticospinal tract involved in differentially regulating motor outputs and sensory inputs. It combines optogenetic stimulation within the sensorimotor cortex of specific corticospinal neurons based on the development of particular intersectional viral strategies with in vivo electrophysiology/ electromyography. They demonstrate that the modulation of sensory inputs from the limb and that of muscle contraction (motor output) are involving different corticospinal pathways. Thus, this paper argues in favor of reconsidering the functions of the corticospinal tract towards a possible additional prominent role in the modulation of sensory inputs. This study is of potential interest to scientists within the field of motor control interested in the organization of corticospinal modulation of sensory inputs.

Essential Revisions:

Three reviewers and I have carefully read the manuscript and came together to the conclusion that while this paper addresses important questions regarding the potential role of the corticospinal tract in regulating motor outputs and sensory inputs through modulation of sensory transmission via primary afferent depolarization (PAD), it contains major flows that must be corrected before being able to judge the suitability for publication in eLife. The three following major concerns must be addressed:

1 – The size of PAD evoked by the opto-stimulation of cortical tract is abnormally small. This could be due to the extracellular recording approach (bad seal of the extracellular electrode), to only a few neurons being transynaptically labelled or to the fact that the CST has negligible effects on PAD (that would be problematic for the interpretations and conclusions reached). Thus the authors must provide data allowing to discriminate between these different hypotheses to also support the conclusion made by the authors. In addition, a positive control measurement of PAD evoked by dorsal root stimulation is lacking. Indeed it would be important to calibrate the PAD size to a known afferent input like dorsal root stimulation.

2 – Anatomical and functional controls must be provided for the different viral infections and CRE-lines used in the study, to make sure that the proper populations are targeted and that this offers the possibility of having a very selective stimulation. The authors must provide rigorous controls of the tracing and optogenetics experiments.

3 – Some experimental series are done using a different anesthesia protocol (ketamine vs isoflurane). It remains possible that this might change the results of the experiments and explain the lack of EMG stimulation. Therefore experiments should be replicated using the same anesthetical agent.

Reviewer #1:

1) The size of the PAD evoked by corticospinal (CS) activation is exceedingly small, 0.5 to 1 uV (not mV), and required 30 – 60 trace averages to see PAD above the noise. This could be because the CST has negligible effects on PAD (which I doubt), or it could be that the recording suction electrodes do not provide a good seal on the dorsal roots and thus much of the signal is lost to extracellular leak currents in the root. This seal can be improved with grease or sucrose, though this is not necessary here (Stys et al. 1993; Huxley and Stampfli, 1951). Instead, at a minimum, it is imperative to compare the authors' CS evoked PAD to classical dorsal root evoked PAD. If they are similar in size then the authors enthusiasm about the CST controlling PAD holds.

2) The Thy-cre animal labels many cell in addition to lamina V CST neurons, and the authors should state this limitation when they introduce the model. This limitation is overcome by the very nice duel virus labelling ChETA insertion model (line 164), since in this case only the CST is labelled. However, the authors only test whether in this model light evokes EMG. It is critical that the authors also show us the light evoked PAD in this ChETA CST model. This will provide direct evidence for CST evoked PAD.

Reviewer #2:

Moreno-Lopez et al., investigated the contribution of the corticospinal tract in motor control and the spinal cord sensory gating. Using tracing tools and in vivo electrophysiology recordings, they mapped the cortical regions responsible for hindlimb muscle contractions and lumbar dorsal root potentials and showed that they are overlapping. They found that the branch of corticospinal tract targeting lumbar interneurons in the spinal cord is involved in mediating dorsal root potentials and by consequence plays a role in the regulation of sensory inputs. On the other hand, the corticospinal tract performs its function in hindlimb motor control via a relay in neurons of the upper spinal cord which then project to the lumbar spinal cord. The data of this manuscript are of great interest to go further in the understanding of the roles that play the corticospinal tract however the lack of rigorous controls of the tracing and optogenetics experiments impede the data to fully support the claims of the authors.

1 – The external stimulation of layer V pyramidal neurons of the sensorimotor cortex in Thy::ChR2 mice seems to elicit both DRPs and EMGs (Figure 1A and B). However, this experiment lacks essential controls to show that the DRP and muscle contraction are not just triggered by the photostimulation of the brain but is really due to the activation of the layer V pyramidal neurons.

The exact same recordings while illuminating the cortex in mice that are not expressing Chr2 is recommended.

It should also be stated on line 80, results, that these mice express ChR2 in other ares of the cortex and brain as well according to Arenkiel et al.

2 – Several statements in this publication rely on the use of the AAV-WGA-CRE tracing from the cortex to the spinal cord. The authors claim that this tracing is anterograde and monosynaptic. However, several publications report that WGA can be bidirectionally transported and can be transmitted polysynaptically (e.g. Levy et al. 2015 Neural Tracing Methods: Tracing Neurons and Their Connections). It seems that the direction of transportation of WGA and its ability to be transsynaptically transmitted depends on the neuronal network. Moreover, they use an AAV2/1 virus and the AAV1 serotype can also be transported in the neurons both anterogradely or retrogradely. Thus, it would be essential to show that the spinal cord traced cells result from the anterograde monosynaptic transmission of the WGA-Cre virus. This is also true for the experiments with ChAT::GFP, in what direction (anterograde, retrograde) do the authors envisage the tracing to take place? An inset in figure supplement 3 would be good to explain this better.

Recommendation: This could be performed using an IHC with an antibody targeting WGA in the spinal cord to prove that the Tomato cells express WGA. If there is any retrograde transport of the WGA-Cre, it might be possible to observe some staining in the afferent tracts in the spinal cord. I would suggest to add a representative picture of the whole spinal cord at the level of the traced neurons in addition to the crop picture in Fig1E. Perhaps a picture of a transverse or sagittal section from the midbrain and/or the brainstem where motor and sensory tracts are well distinct could also confirm that there is no staining in the sensory ascending tracts.

3 – There is a large disparity in the number of td-tom cells that were counted in GAD65::GFP and ChaT::GFP (427 neurons in GAD65::GFP from 3 mice and 45 in ChaT::gfp from 4 mice). Why such a disparity? Were there less cells in the ChaT::GFP tracing or did the authors just count less spinal cord sections?

It would be interesting to know the average number of Tomato neurons traced in the spinal cord and if this number is consistent between animals.

4 – The conclusion from Figure1 is that most of the spinal cord neurons targeted by CST are excitatory. Direct evidence that most of these neurons are really glutamatergic would reinforce the data. One possibility would be to perform in situ hybridization with a Vglut2 probe.

5 – There is a large difference in the intensity of the DRPs between Figure 1 (between 20 and 30uV) and Figure 2 (3.8uV). Why?

6 – The optogenetic stimulation performed in Figure 3 lacks a similar control as the one performed in Figure 1.

7 – In Figure 4. the spinal cord optogenetic experiments lack controls to check that the light by itself does not affect the recordings.

The neurons targeted by the optogenetic stimulation are located deep in the dorsal horn. Few in vivo spinal cord optogenetic stimulation experiments have been published so far. Thus, little evidence of the reliability of such experiments is available and a control showing that these neurons are indeed activated by the light stimulation would strengthen the data.

WGA-Cre was injected in the brain and is by consequence expressed as well in the corticospinal tract. Is there any ChETA-eYFP expression in the CST?

How many spinal cord neurons were ChETA-eYFP positive?

For this particular experiment, the authors had to switch between ketamine and isoflurane anesthesia. Could the lack of EMG stimulation be a consequence of this different anesthesia protocol?

Recommendation: To reproduce the spinal cord optogenetic stimulation experiment in animals that do not express Chr2 (ideally with a control virus injected). To check for the activation of the spinal cord neurons, for example, an immunohistochemistry against c-fos could be performed after light stimulation. Add a representative picture of the whole spinal cord to show that there is no expression of CHETA-eYFP in the corticospinal tract.

Further, to demonstrate that the particular anesthesia protocol used for this experiment is not the cause of the absence of muscle contraction, the same anesthesia protocol while performing the same optogenetic stimulation experiment shown in Figure 3 should be done (retro AAV-ChR2-mcherry in the spinal cord and light stimulation of the cortex).

Reviewer #3:

This study combines optogenetic recruitment of corticospinal tract neurons within mouse sensorimotor cortex, differentiated from other indirect corticofugal pathways via pyramidotomy, with various AAV-based transgene delivery approaches to compare and contrast corticospinal presumed GABAergic presynaptic inhibitory actions on unidentified hindlimb primary afferents in lumbar cord (measured as dorsal root potentials as an index of primary afferent depolarization) versus recruitment of tibialis anterior ankle flexor motor neurons (measured electromyographically).

The use of AAV cortical delivery of genetic construct generating WGA-Cre fusion protein in double transgenic Cre-dependent tdTomato/GAD65-GFP mice or in Cre-dependent tdTomato mice with additional intraspinal AAV Cre-dependent CHR2 variant (CHETA) delivery provided powerful molecular tools to demonstrate; (i) that corticospinal neurons projected to a small but significant fraction of GAD65 last-order inhibitory interneurons as well as many interposed putative excitatory interneurons involved in generating primary afferent depolarization (PAD), and (ii) that a distributed albeit small population of predominantly corticospinal tract (CST)-based transsynaptic optogenetically-labeled/recruited deep dorsal horn interneurons are capable of generating PAD. These are important and compelling observations.

The major conclusions are that sensorimotor cortical control of spinal hindlimb presynaptic afferent input via PAD is by corticospinal actions onto lumbar interneurons yet activation of a similar cortical region instead controls motor excitability via cortical projections to brainstem descending and/or more rostral spinal regions. However, one cannot exclude the possibility that differentiable actions may also arise due to use of different optogenetic cortical stimulation paradigms for studies on sensory input versus motor output systems. No explanation or rationale is given for the use of illumination area and stimulus train differences in studies on motor versus sensory function.

The anatomical/molecular studies are elegant and well described. It would have been helpful if anatomical studies included use of additional fluorescent immunolabeling To further identify transsynaptic WGA labeled interneurons as being excitatory (figure 1) or inhibitory (figure 4).

Overall, the work presents important and compelling observations on the organization of corticospinal projections onto interneurons in the lumbar spinal cord and distinct pathways involved in cortical control of sensory input versus motor output. Derived insights will help guide future studies on the organization of corticospinal modulation of sensory input.

The greatest weakness of the paper is the inability to study cortical recruitment of motor output (via EMG) and afferent input (via recorded DRPs in a dorsal root) simultaneously in the same animals under the same photostimulation (and anesthetic) protocols. There were differences in (i) photostimulation probes sizes (250 vs 105 μm), (ii) stimulus paradigms, (iii) anesthetics (ketamine vs isoflurane), and likely different optical power (laser vs LED) applied to assess lumbar spinal motor versus sensory function, respectively. How these variables bias neuronal in pathway recruitment was not considered. While use of different anesthetics may have been justifiably argued on ethical grounds, it unfortunately does not detract from the possibility that such differences may differentially alter neuronal recruitment, pathways and circuit excitability.

The work presents important and compelling observations on the organization of corticospinal projections onto interneurons in the lumbar spinal cord and distinct pathways involved in cortical control of sensory input versus motor output. Derived insights will help guide future studies on the organization of corticospinal modulation of sensory input.

This study combines optogenetic recruitment of corticospinal tract neurons within mouse sensorimotor cortex, differentiated from other indirect corticofugal pathways via pyramidotomy, with various AAV-based transgene delivery approaches to compare and contrast corticospinal presumed GABAergic presynaptic inhibitory actions on unidentified hindlimb primary afferents in lumbar cord (measured as dorsal root potentials as an index of primary afferent depolarization) versus recruitment of tibialis anterior ankle flexor motor neurons (measured electromyographically).

The use of AAV cortical delivery of genetic construct generating WGA-Cre fusion protein in double transgenic Cre-dependent tdTomato/GAD65-GFP mice or in Cre-dependent tdTomato mice with additional intraspinal AAV Cre-dependent CHR2 variant (CHETA) delivery provided powerful molecular tools to demonstrate; (i) that corticospinal neurons projected to a small but significant fraction of GAD65 last-order inhibitory interneurons as well as many interposed putative excitatory interneurons involved in generating primary afferent depolarization (PAD), and (ii) that a distributed albeit small population of predominantly corticospinal tract (CST)-based transsynaptic optogenetically-labeled/recruited deep dorsal horn interneurons are capable of generating PAD. These are important and compelling observations.

The major conclusions are that sensorimotor cortical control of spinal hindlimb presynaptic afferent input via PAD is by corticospinal actions onto lumbar interneurons yet activation of a similar cortical region instead controls motor excitability via cortical projections to brainstem descending and/or more rostral spinal regions. However, one cannot exclude the possibility that differentiable actions may also arise due to use of different optogenetic cortical stimulation paradigms for studies on sensory input versus motor output systems. No explanation or rationale is given for the use of illumination area and stimulus train differences in studies on motor versus sensory function.

The anatomical/molecular studies are elegant and well described. It would have been helpful if anatomical studies included use of additional fluorescent immunolabeling To further identify transsynaptic WGA labeled interneurons as being excitatory (figure 1) or inhibitory (figure 4).

The greatest weakness of the paper is the inability to study cortical recruitment of motor output (via EMG) and afferent input (via recorded DRPs in a dorsal root) simultaneously in the same animals under the same photostimulation (and anesthetic) protocols. There were differences in (i) photostimulation probes sizes (250 vs 105 μm), (ii) stimulus paradigms, (iii) anesthetics (ketamine vs isoflurane), and likely different optical power (laser vs LED) applied to assess lumbar spinal motor versus sensory function, respectively. How these variables bias neuronal in pathway recruitment was not considered. While justification for anesthetic differences may have been justifiably argued on ethical grounds, it unfortunately does not detract from the possibility that such differences may differentially alter neuronal recruitment, pathways and circuit excitability. It seems like experiments that record DRPs also recording EMG would be helpful to discriminate differences.

It may be helpful to undertake some simple experiments in Thy1::ChR2 mice to explore use of an anesthetic that permit simultaneous study of effects of cortical optogenetic stimulation on both sensory and motor systems in the same animal using identical photostimulation parameters, as well as for back-comparison to the different parameters used in the presently presented separate studies on corticospinal control of motor and sensory transmission. An anesthetic to consider is injectable long-lasting urethane anesthesia (Maggi and Meli 1986: 10.1007/bf01952426) which is only allowed for non-survival surgery that has been used in many studies on PAD (e.g. Lidierth 2006 – J Physiology) and likely has robust EMG activity (e.g. Zhang, C., et al., (2018) DOI: 10.5213/inj.1835052.52). While veterinary approval of urethane use is discouraged due to carcinogenic potential, use of protective clothing should easily justify its use to vetrinary staff given it is a very reliable and effective anesthetic with strong scientific rationale.

Although I fully appreciate the difficulty of the experiments, more insight on organization could have been provided using limited additional electrophysiological approaches for characterization of (i) motor and (ii) sensory pathways. (i) EMG recordings are rather easy in terminal experimentation, so it would have been helpful to know whether the cortical stimulation site chosen to recruit distal limb musculature (the foot flexor tibialis anterior) had somatotopic selectivity by also recording from a knee and hip flexor muscle. This could be incorporated into the additional urethane experiments suggested above. (ii) Use of an additional dorsal root or peripheral nerve for electrical stimulation in condition-test paradigms with CST photostimulation could have verified the expectation that corticospinal modulation of PAD was on interneurons interposed in low threshold afferent pathways (as characterized in the cat by Rudomin and colleagues) or when C-fibers as shown by this group in rat in one of their earlier studies (Moreno-López, Y., et al., (2013). PLoS One 8(7): e69063).

eLife. 2021 Sep 9;10:e65304. doi: 10.7554/eLife.65304.sa2

Author response


Essential revisions

Three reviewers and I have carefully read the manuscript and came together to the conclusion that while this paper addresses important questions regarding the potential role of the corticospinal tract in regulating motor outputs and sensory inputs through modulation of sensory transmission via primary afferent depolarization (PAD), it contains major flows that must be corrected before being able to judge the suitability for publication in eLife. The three following major concerns must be addressed:

1 – The size of PAD evoked by the opto-stimulation of cortical tract is abnormally small. This could be due to the extracellular recording approach (bad seal of the extracellular electrode), to only a few neurons being transynaptically labelled or to the fact that the CST has negligible effects on PAD (that would be problematic for the interpretations and conclusions reached). Thus the authors must provide data allowing to discriminate between these different hypotheses to also support the conclusion made by the authors. In addition, a positive control measurement of PAD evoked by dorsal root stimulation is lacking. Indeed it would be important to calibrate the PAD size to a known afferent input like dorsal root stimulation.

We acknowledge the reviewers concerns about the size of the cortically-evoked dorsal root potentials (DRPs). We did some additional experiments presented below but we would like here to stress two important points. First, we are recording dorsal root potentials, not directly PAD. While PAD yields to deflections in the (low) mV range (recorded intra-axonally), DRPs are classically in the μV range, as observed originally in the Cat by Gossard and collaborators performing both these approaches (Wall, 1994). DRP recordings have rarely been performed in mice, and one of the few articles presenting them illustrate signals in the 100-150 μV range (Grunewald and Geis, 2014).

Second, as well noted by the reviewing editor above, the size of the DRP depends on the source eliciting it. The mouse study by Grunewald and Geis only presents segmentally-evoked DRPs. A seminal article by Wall and Lidierth (Wall and Lidierth, 1997) demonstrates that, in Rats, Cx-evoked DRPs are approximatively of 25μV amplitude, while segmental DRP are much larger, several hundreds of μV. Large segmental DRPs are also observed in the original publication by the same author (Wall, 1994).

We have now provided a direct measure of the size of the DRP evoked by the opto-stimulation of the cortical tract, independent of the transynaptic experiment. We have performed DRP recordings in mice where CS neurons are retrogradelly infected by a ChR2-encoding virus (Figure 3 —figure supplement 1). We also present in this figure the individual DRP amplitudes for all the recordings performed in the study, i.e. in three different mouse models, means presented in Author response table 1.

Author response table 1.

Mouse model Stimulation site N Mean DRP ampl. Range DRP ampl.
Thy1 mice Cortex 10 7.0 μV 3.3 μV – 20.4 μV
ChR2 in CS targets Spinal Cord 8 2.9 μV 0.9 μV – 5.0μV
ChR2 in CS neurons Cortex 5 14.5 μV 8.4 μV – 26.8 μV

The DRPs amplitude recorded when the CS is exclusively stimulated is in the same range as the one recorded when the sensorimotor cortex is stimulated, and to similar study performed in Rats (Wall and Lidierth, 1997). It is indeed smaller when only the targets of the CS are stimulated in the spinal cord, as this approach is not expected to stimulate all of the targets of the CST. We did however try to perform segmental PAD recordings, but we were limited by our equipment that prevented the proper placement of the required electrodes in such a small available space in mice (in addition to the spinal clamps).

A last point we would like to mention is that a small deflection in the μV range does not mean that the physiological effect is negligible. Indeed, recordings in the monkey by Seki and collaborators has shown effective sensory presynaptic inhibition correlated with a modest (<2μV) change in the amplitude of the antidromic response recorded in the afferent nerve (Seki et al., 2003). This is now included in the discussion (l. 345-349).

2 – Anatomical and functional controls must be provided for the different viral infections and CRE-lines used in the study, to make sure that the proper populations are targeted and that this offers the possibility of having a very selective stimulation. The authors must provide rigorous controls of the tracing and optogenetics experiments.

We agree that the anatomical and functional controls presented in the original version of the manuscript were incomplete and we now provide additional ones in supplementary figures. These include :

– Lack of labelling in the spinal cord ascending tract or in the cerebellum deep nuclei after AAV WGA-Cre injection in the cortex, demonstrating that spinal TdTomato neurons have been labeled through transynaptic anterograde tracing (Figure 1 —figure supplement 2).

– Appropriate dorsal horn view to show that the CST does not target dorsal horn cholinergic interneurons (Figure 1 —figure supplement 3).

– DRP recordings after selective stimulation of CS neurons (after their retrograde infection by spinal injection of rAAV ChR2) (Figure 3 —figure supplement 1).

– Absence of EMG and DRP signal after cortical stimulation of GFP-expressing CS neurons (Figure 3 —figure supplement 1).

– Absence of correlation between the number of CS targets transynaptically labeled and the survival time or the cortical area infected (Figure 4 —figure supplement 1).

– Absence of labelling of the CST after the intersectional approach to induce ChETA expression in the lumbar CS targets (Figure 4 —figure supplement 2).

– Labeling of ventral horn neurons (corresponding to the deepest targets of the CS) with the intraspinal AAV ChETA-floxed injection (Figure 4 —figure supplement 2).

– Analysis of the light penetration after surface illumination of the dorsal horn: this photostimulation induced muscle contraction in Thy1::ChR2 animals, and directly activates neurons located as deep as 600 μm (single unit recordings) (Figure 4 —figure supplement 3).

3 – Some experimental series are done using a different anesthesia protocol (ketamine vs isoflurane). It remains possible that this might change the results of the experiments and explain the lack of EMG stimulation. Therefore experiments should be replicated using the same anesthetical agent.

We aimed at performing the two types of recordings (EMG and DRP) with the same anesthetical agent, unfortunately we do not believe it is possible (see below) and, most importantly, we do not believe it biases the result of the present study nor explains the lack of EMG signal after spinal photostimulation.

We are thankful to the reviewers for their suggestions to try and identify unifying recording conditions. Unfortunately recording cortically-evoked EMGs requires even more stringent conditions than spinally evoked-ones. For example, we now show (Figure 4 —figure supplement 3) that surface stimulation of the spinal cord induces EMG signal in Thy1::ChR2 animals under isoflurane anesthesia, but this was never the case after cortical stimulation. Similarly, we were not able to obtain Cx-EMG under urethane anesthesia in preliminary experiments. Ketamine, anesthesia that was the only condition where we obtained Cx-EMG; but this anesthesia was too light to be compatible with the large laminectomy required for DRP recordings (procedure refused by our ethical committee).

Importantly, we do not compare responses thresholds nor the extent of the cortical area producing a response in between types of recordings (EMG vs. DRP). Throughout the manuscript, we base our conclusions on comparisons of a given signal (EMG or DRP), with a given experimental approach, in different animal models (THY1, ChR2-retro, pyramidotomy, transynaptically labeled CS targets). These conclusions are therefore not impacted by the differences in recording conditions between EMGs and DRPs.

However, we have now expanded the “reliability of circuit investigations” section to further discuss the consequences of obtaining results with different anesthesia (and stimulation conditions in general).

Lastly, concerning the lack of EMG signal after local stimulation of the CST targets, we now provide additional arguments to rule out experimental biases. As explained below in the individual responses, we have previously perform a similar switch of anesthesia without impact on EMG responses. We also provide anatomical and functional controls demonstrating that our intraspinal AAV injections can reach the deepest CS targets and that surface illumination of the cord also penetrates enough to activate them.

Reviewer #1:

1) The size of the PAD evoked by corticospinal (CS) activation is exceedingly small, 0.5 to 1 uV (not mV), and required 30 – 60 trace averages to see PAD above the noise. This could be because the CST has negligible effects on PAD (which I doubt), or it could be that the recording suction electrodes do not provide a good seal on the dorsal roots and thus much of the signal is lost to extracellular leak currents in the root. This seal can be improved with grease or sucrose, though this is not necessary here (Stys et al. 1993; Huxley and Stampfli, 1951). Instead, at a minimum, it is imperative to compare the authors' CS evoked PAD to classical dorsal root evoked PAD. If they are similar in size then the authors enthusiasm about the CST controlling PAD holds.

This point was raised by the reviewing editor and our response is to be found in the first page of this document.

2) The Thy-cre animal labels many cell in addition to lamina V CST neurons, and the authors should state this limitation when they introduce the model. This limitation is overcome by the very nice duel virus labelling ChETA insertion model (line 164), since in this case only the CST is labelled.

The expression in other cells is now stated when presenting this mouse line (l. 99-100). The limitations are indeed presented when introducing the retrograde virus model.

However, the authors only test whether in this model light evokes EMG. It is critical that the authors also show us the light evoked PAD in this ChETA CST model. This will provide direct evidence for CST evoked PAD.

We have now included DRP measurements after exclusive stimulation of CS neurons (from retrograde viral infection of a ChR2 encoding virus in the spinal cord). Stimulation of CS neurons indeed induced DRPs and this is now presented in supplementary Figure 3 —figure supplement 1.

Reviewer #2:

1 – The external stimulation of layer V pyramidal neurons of the sensorimotor cortex in Thy::ChR2 mice seems to elicit both DRPs and EMGs (Figure 1A and B). However, this experiment lacks essential controls to show that the DRP and muscle contraction are not just triggered by the photostimulation of the brain but is really due to the activation of the layer V pyramidal neurons.

The exact same recordings while illuminating the cortex in mice that are not expressing Chr2 is recommended.

We now include in supplementary Figure 3—figure supplement 1 the recordings obtained from animals injected with an EGFP-encoding retro-AAV in the spinal cord, where photostimulation of the cortex leads to no DRP or EMG signal. This control is now mentioned in l. 104-105 and 171-173.

It should also be stated on line 80, results, that these mice express ChR2 in other ares of the cortex and brain as well according to Arenkiel et al.

This is now indicated.

2 – Several statements in this publication rely on the use of the AAV-WGA-CRE tracing from the cortex to the spinal cord. The authors claim that this tracing is anterograde and monosynaptic. However, several publications report that WGA can be bidirectionally transported and can be transmitted polysynaptically (e.g. Levy et al., 2015 Neural Tracing Methods: Tracing Neurons and Their Connections). It seems that the direction of transportation of WGA and its ability to be transsynaptically transmitted depends on the neuronal network.

The tracing properties of WGA seem to depend on the mode of injection/expression. When the lectin itself is injected, it can indeed travel in the retrograde direction (LeVay and Voigt, 1990). However the seminal studies using WGA-expressing transgenic mice already mention an exclusive anterograde transneuronal tracing (Braz et al., 2002). This was later confirmed by several other groups, including for the specific WGA-Cre fusion AAV construct (Gradinaru et al., 2010; Libbrecht et al., 2017). One puzzling exception is the study by Xu and Südhof (Xu and Sudhof, 2013) where WGA-Cre (encoded in AAV) is presented as a transynaptic retrograde tracer. In many cases, regions are reciprocally connected and it is difficult to conclude unequivocally on the direction of the transfer. In Suppl. Figure 4 of the Xu and Südhof paper, for example, they show axons of primarily infected neurons (N. Reuniens neurons) in the cortex, next to transynaptically labelled cortical neurons. One could interpret that the cortical neurons have been labelled through transynaptic anterograde passage of the WGA-Cre from the WGA-Cre expressing axons in their very close vicinity; yet the authors conclude that the cortical neurons received WGA-Cre by retrograde tracing of cortico-reuniens neurons.

Concerning our study, the important point is to be sure that spinal labeled neurons are indeed direct targets of the cortex. As suggested by the reviewer below, we can exclude a retrograde tracing because spinal ascending tracts are devoid of labelling (Figure 1 —figure supplement 2). In the same figure, we also illustrate deep cerebellar nuclei, that are, like the spinal cord, two synapses away in the retrograde direction, and that show no labelling.

More generally, in contrast with a transynaptic virus that can be amplified in the target neuron (and then cross another synapse), it is the WGA-Cre fusion protein that is transported transneuronally and thus is highly diluted. For another study, we have used an AAV encoding for the WGA-EGFP fusion protein: even with long survival times, the EGFP was never visible in the target neurons without immunohistochemical amplification, suggesting low concentration after transynaptic transfer. Using the WGA-Cre constructs (in TdTomato-floxed mice) ensures that the synaptic targets can be identified without further amplification. However we, and the above cited studies, see no evidence of multiple synapse crossing. Some elements of this response are now included in Figure 1 —figure supplement 2.

Moreover, they use an AAV2/1 virus and the AAV1 serotype can also be transported in the neurons both anterogradely or retrogradely. Thus, it would be essential to show that the spinal cord traced cells result from the anterograde monosynaptic transmission of the WGA-Cre virus.

The WGA-Cre AAV1 virus is injected in the cortex. To explain the presence of Cre-expressing neurons in the spinal cord as the consequence of retrograde transport, we should hypothesize a retrograde transynaptic labeling, as there is no direct spino-cortical tract. As stated in the above response, we now include in Figure 1 —figure supplement 2 several elements demonstrating that the spinal labeling does not result from transynaptic retrograde transport.

This is also true for the experiments with ChAT::GFP, in what direction (anterograde, retrograde) do the authors envisage the tracing to take place? An inset in figure supplement 3 would be good to explain this better.

We thank the reviewer for this suggestion and have now included an inset in the figure to illustrate the anterograde transynaptic labeling in ChAT::EGFP mice. The demonstration of anterograde transport is the same as above as it is the same tool.

Recommendation: This could be performed using an IHC with an antibody targeting WGA in the spinal cord to prove that the Tomato cells express WGA. If there is any retrograde transport of the WGA-Cre, it might be possible to observe some staining in the afferent tracts in the spinal cord. I would suggest to add a representative picture of the whole spinal cord at the level of the traced neurons in addition to the crop picture in Fig1E. Perhaps a picture of a transverse or sagittal section from the midbrain and/or the brainstem where motor and sensory tracts are well distinct could also confirm that there is no staining in the sensory ascending tracts.

We are thankful for this suggestion that elegantly rules out retrograde labeling at the level of the spinal cord (which, again, does not project directly to the somatosensory cortex). We have now included a larger view of the spinal cord in Figure 1 —figure supplement 2, showing that labeling is restricted to the ventral part of the dorsal funiculus (where the dorsal corticospinal tract resides) and in the grey matter.

3 – There is a large disparity in the number of td-tom cells that were counted in GAD65::GFP and ChaT::GFP (427 neurons in GAD65::GFP from 3 mice and 45 in ChaT::gfp from 4 mice). Why such a disparity? Were there less cells in the ChaT::GFP tracing or did the authors just count less spinal cord sections?

It would be interesting to know the average number of Tomato neurons traced in the spinal cord and if this number is consistent between animals.

We did a systematic counting in all animals and numbers are now included in Figure 4 – Supplementary Figure 1. There was a large variation in the numbers of transynaptic labeled neurons that was not correlated to survival time post-injection, nor to the size of the infected cortical area (this is now detailed in the suppl. Figure).

4 – The conclusion from Figure1 is that most of the spinal cord neurons targeted by CST are excitatory. Direct evidence that most of these neurons are really glutamatergic would reinforce the data. One possibility would be to perform in situ hybridization with a Vglut2 probe.

This conclusion required more literature support, as also pointed out by reviewer 1. Although we agree that a better characterization of the neurochemical nature of CS targets would be very interesting, the proposed in situ hybridization (on top of the transynaptic tracing) was beyond our present expertise. We thus took out this sentence as the important conclusion of this experiment is that some GAD65 neurons are directly targeted by the CST.

5 – There is a large difference in the intensity of the DRPs between Figure 1 (between 20 and 30uV) and Figure 2 (3.8uV). Why?

In addition to the plot now included in Figure 3—figure supplement 1, presenting DRP amplitudes, we present in Author response image 1 an additional dissection of the results depending on the type of experiment and on the experimenter. It so happens that the few animals on which the pyramidotomy was performed (n=3, THY1 prePYR in Author response image 1, and also in Figure 2) are among the THY1 animals where the smallest DRP amplitudes were recorded. The DRP amplitude does not depend so much on the experimenter, but rather on the quality of the dissection and size of the rootlet.

Author response image 1. DRP amplitude as a function of experiment type and experimenter.

Author response image 1.

THY1: experiments on THY1-ChR2 animals (Fig. 1), THY1 prePYT: experiments on THY1-ChR2 where a pyramidotomy was performed (Fig. 2), measures taken before the pyramidotomy, Retro: animals injected with ChR2-retro AAV in the spinal cord (Fig 3-Figure supplement 1), N+1: animals with a double viral injection inducing ChETA expression in the CS lumbar targets (Fig. 4). The first three are responses to cortical photostimulations, the last one corresponds to responses to spinal photostimulations.YML, CB, GD: the three experimenters that performed the recordings

Also, the group of animals where ChR2 in expressed in CS targets (named “N+1” in Author response image 1) repeatedly demonstrated a lower amplitude of DRPs. This was expected, as the efficacy of transynaptic transfer was limited; thus, only a fraction of the total CS targets could be activated.

6 – The optogenetic stimulation performed in Figure 3 lacks a similar control as the one performed in Figure 1.

This control is now included in suppl. Figure 3 —figure supplement 1.

7 – In Figure 4. the spinal cord optogenetic experiments lack controls to check that the light by itself does not affect the recordings.

Photostimulation of the spinal cord induced induced DRPs in the ipsilateral lumbar root of 8 out of 9 mice. In one mouse, the same photostimulation induced no significant DRP demonstrating that the light itself does not produce an artefact that we would misinterpret as a response.

The neurons targeted by the optogenetic stimulation are located deep in the dorsal horn. Few in vivo spinal cord optogenetic stimulation experiments have been published so far. Thus, little evidence of the reliability of such experiments is available and a control showing that these neurons are indeed activated by the light stimulation would strengthen the data.

We already mentioned in the discussion (l. 265) that we were able to record an intraspinal LFP as deep as 1150 μm after photostimulating the surface of the cord, in the double infected (ChETA in the CS targets) mice.

To directly address this issue with our experimental configuration, we performed additional controls in THY1 animals, now included in a new Figure 4 —figure supplement 3 (and described in l. 208-212). These experiments illustrate that surface illumination induces muscle contraction (and EMG signal, even under isoflurane anesthesia) as well as direct activation of ChR2-expressing ventral horn neurons. This demonstrates that the light efficiently penetrates the cord as deep as 600μm after surface illumination.

Finally, for spinal photostimulations we used our largest probe (1,1 mm) at a power of 42 mW/cm2. With powers equal or lower than this, and a similar surface illumation, Caggiano and colleagues were able to induce motor contraction in THY1 and ChAT-ChR2 animals (Caggiano et al., 2016); in the latter experiment, they consider stimulating directly motoneurons located in the deep ventral horn.

WGA-Cre was injected in the brain and is by consequence expressed as well in the corticospinal tract. Is there any ChETA-eYFP expression in the CST?

We now include a picture (Figure 4 —figure supplement 2) illustrating the absence of ChETA-eYFP expression in the CST. This demonstrates that the intraspinal injection of AAV1-Flex-ChETA does not lead to retrograde infection of CS neurons, and that only the lumbar targets of the CST express ChETA in this intersectional experiment. This is described in the main text on l. 204-206.

How many spinal cord neurons were ChETA-eYFP positive?

The numbers are now indicated in Figure 4 —figure supplement Figure 2. As stated there, these might be underestimated as the histological analysis was performed after a long recording session, with only post-fixation (no intracardiac perfusion of fixative).

For this particular experiment, the authors had to switch between ketamine and isoflurane anesthesia. Could the lack of EMG stimulation be a consequence of this different anesthesia protocol?

In another project, we routinely perform isoflurane to ketamine/xylazine switches of anesthesia, without compromising EMG responses, we thus do not believe that this might be an explanation for the lack of EMG response.

Recommendation: To reproduce the spinal cord optogenetic stimulation experiment in animals that do not express Chr2 (ideally with a control virus injected). To check for the activation of the spinal cord neurons, for example, an immunohistochemistry against c-fos could be performed after light stimulation.

We are not sure about the point the reviewer is willing to raise here. We now show a control (single unit recording) of a ventral horn neuron activated by the surface illumination. Is this answering the reviewer’s point? We also mentioned earlier that we did not observe DRP signal in 1 out of 9 mice, demonstrating that the signal we interpret as DRP is not produced by the light alone.

Add a representative picture of the whole spinal cord to show that there is no expression of CHETA-eYFP in the corticospinal tract.

We now include a picture of the whole spinal cord (Figure 4 —figure supplement 2) illustrating the absence of ChETA-eYFP expression in the CST.

Further, to demonstrate that the particular anesthesia protocol used for this experiment is not the cause of the absence of muscle contraction, the same anesthesia protocol while performing the same optogenetic stimulation experiment shown in Figure 3 should be done (retro AAV-ChR2-mcherry in the spinal cord and light stimulation of the cortex).

EMGs are always recorded in ketamine/xylazine. In this particular experiment (ChETA in the targets of the CST), we switch between isoflurane and ketamine/xylazine, but as mentioned above, this is a protocol we have used in another project without negatively impacting EMG measures.

Reviewer #3:

The major conclusions are that sensorimotor cortical control of spinal hindlimb presynaptic afferent input via PAD is by corticospinal actions onto lumbar interneurons yet activation of a similar cortical region instead controls motor excitability via cortical projections to brainstem descending and/or more rostral spinal regions. However, one cannot exclude the possibility that differentiable actions may also arise due to use of different optogenetic cortical stimulation paradigms for studies on sensory input versus motor output systems. No explanation or rationale is given for the use of illumination area and stimulus train differences in studies on motor versus sensory function.

The two types of study involve stimulation of the same area: either mapping of the sensorimotor cortex for Figure 1, or a stimulation located at the center of the two maps for the following cortical stimulations. We have now included a reference to the articles that inspired our stimulation protocols (Methods l. 456-458).

The anatomical/molecular studies are elegant and well described. It would have been helpful if anatomical studies included use of additional fluorescent immunolabeling To further identify transsynaptic WGA labeled interneurons as being excitatory (figure 1) or inhibitory (figure 4).

Although we agree that a better characterization of the neurochemical nature of CS targets would be very interesting, reliable antibodies labeling large subpopulations of dorsal horn neurons are scarce, and most studies rely on the use of transgenic reporter mouse to identify, for example, excitatory neurons. We could test GAD65 mice which were important in our project as GAD65+ neurons are known to form presynaptic contacts onto proprioceptive terminals; we could thus demonstrate that some GAD65 neurons were directly targeted by the CST. Further characterization would require new tools that are beyond the scope of the present study.

The greatest weakness of the paper is the inability to study cortical recruitment of motor output (via EMG) and afferent input (via recorded DRPs in a dorsal root) simultaneously in the same animals under the same photostimulation (and anesthetic) protocols. There were differences in (i) photostimulation probes sizes (250 vs 105 μm), (ii) stimulus paradigms, (iii) anesthetics (ketamine vs isoflurane), and likely different optical power (laser vs LED) applied to assess lumbar spinal motor versus sensory function, respectively. How these variables bias neuronal in pathway recruitment was not considered.

We agree with the reviewer that many parameters differentiate the two types of recordings, and some of the consequences were already discussed in the “reliability of circuit investigations” section. We have now developed this section with a discussion on the recruitment threshold (l. 243-247). We do not believe that this will bias our results as we do not compare thresholds in between stimulation/recording configurations, but rather analyze the amplitude of a given signal (EMG or DRP), with a given experimental approach, in different animal models (THY1, ChR2-retro, pyramidotomy, transynaptically labeled CS targets).

While use of different anesthetics may have been justifiably argued on ethical grounds, it unfortunately does not detract from the possibility that such differences may differentially alter neuronal recruitment, pathways and circuit excitability.

It may be helpful to undertake some simple experiments in Thy1::ChR2 mice to explore use of an anesthetic that permit simultaneous study of effects of cortical optogenetic stimulation on both sensory and motor systems in the same animal using identical photostimulation parameters, as well as for back-comparison to the different parameters used in the presently presented separate studies on corticospinal control of motor and sensory transmission. An anesthetic to consider is injectable long-lasting urethane anesthesia (Maggi and Meli 1986: 10.1007/bf01952426) which is only allowed for non-survival surgery that has been used in many studies on PAD (e.g. Lidierth 2006 – J Physiology) and likely has robust EMG activity (e.g. Zhang, C., et al., (2018) DOI: 10.5213/inj.1835052.52). While veterinary approval of urethane use is discouraged due to carcinogenic potential, use of protective clothing should easily justify its use to vetrinary staff given it is a very reliable and effective anesthetic with strong scientific rationale.

Obtaining an EMG response after cortical stimulation requires a specific and very light anesthesia. In the Zhang et al., paper, the EMGs obtained in urethane-anesthetized mice were not evoked by supraspinal stimulations. We have similarly obtained EMG signals after spinal stimulation (in THY1 animals) under light isoflurane stimulation (now illustrated in Figure 4 —figure supplement 3). However, we have unsuccessfully attempted to evoke EMG from cortical stimulation under urethane anesthesia in initial exploratory experiments.

Although I fully appreciate the difficulty of the experiments, more insight on organization could have been provided using limited additional electrophysiological approaches for characterization of (i) motor and (ii) sensory pathways. (i) EMG recordings are rather easy in terminal experimentation, so it would have been helpful to know whether the cortical stimulation site chosen to recruit distal limb musculature (the foot flexor tibialis anterior) had somatotopic selectivity by also recording from a knee and hip flexor muscle. This could be incorporated into the additional urethane experiments suggested above.

Although EMG recordings per se are not a difficult experimentation, obtaining a somatotopic map is very demanding because there is only a small window of time during which stimulations/recordings can be performed: the ketamine/xylazine anesthesia has to be light enough to enable movements, but without risking awakening of the animal. In practice, the experimenter had only a few seconds before an additional anesthesia dose was required, that just allowed to obtain the 3-4 replicates needed for a specific coordinate. Building the cortical map thus required multiple re-injection of anesthetics and we observed some tolerance that limited the duration of the experiment.

Our results perfectly coincided with studies published earlier by (Tennant et al., 2011). As they describe the full somatotopy of the hindlimb sensorimotor cortex, and because of the aforementioned difficulty in obtaining a map, we did not attempt to further reproduce these data.

(ii) Use of an additional dorsal root or peripheral nerve for electrical stimulation in condition-test paradigms with CST photostimulation could have verified the expectation that corticospinal modulation of PAD was on interneurons interposed in low threshold afferent pathways (as characterized in the cat by Rudomin and colleagues) or when C-fibers as shown by this group in rat in one of their earlier studies (Moreno-López, Y., et al., (2013). PLoS One 8(7): e69063).

We agree that elucidating the type of sensory modality modulated by the CS tract would be an important advance in the field, however at this stage we consider this as an interesting follow-up project requiring the development of specific expertise and technical approaches for it to be addressed in mice. It is mentioned in the perspectives of our discussion (l. 353-355).

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Moreno-Lopez Y, Bichara C, Delbecq G, Isope P, Cordero-Erausquin M. 2021. The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord (Raw data) Zenodo. [DOI] [PMC free article] [PubMed]

    Supplementary Materials

    Transparent reporting form

    Data Availability Statement

    All raw data for each figure have been made available on Zenodo.

    The following dataset was generated:

    Moreno-Lopez Y, Bichara C, Delbecq G, Isope P, Cordero-Erausquin M. 2021. The corticospinal tract primarily modulates sensory inputs in the mouse lumbar cord (Raw data) Zenodo.


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