Activation of hypothalamic-pontine-spinal pathway promotes locomotor initiation and functional recovery after spinal cord injury in mice

Domain: physics
Published: 2025-12-06
Editorial outcome: major_revision

Abstract
The hypothalamus is critical for regulating behaviors essential for survival and locomotion, but how it integrates internal needs and transmits locomotion commands to the spinal cord (SC) remains unclear. We found that glutamatergic neurons in lateral hypothalamic area (LHA) are essential for regulating motivated locomotor activity. Using single-neuron projectome analysis, trans-synaptic tracing, and optogenetic manipulation, we showed that LHA facilitates motivated locomotion during food seeking via pontine reticular nucleus, oral part (PnO) projection neurons, rather than direct SC projections or indirect stress signaling via medial septum and diagonal band. Activating PnO-SC projection neurons also initiated locomotion. Importantly, LHA-PnO projection neurons were crucial for regulating locomotor recovery following mouse spinal cord injury (SCI). Motor cortex signals gated LHA deep brain stimulation treatment markedly promoted long-term restoration of hindlimb motor functions after severe SCI. Thus, we have identified a hypothalamic-pontine-spinal pathway and the stimulation paradigm for potential therapeutic intervention after SCI.

Introduction
Locomotion is a fundamental behavior that is essential for survival, as it enables animals to seek food, evade threats, and engage in social and reproductive activities. Two primary command centers in the brain, the subthalamic locomotor region (SLR) and the mesencephalic locomotor region (MLR), are responsible for transmitting locomotor commands to the central pattern generators in the spinal cord1–4. Extensive studies have revealed the role of the MLR in controlling locomotion speed and various aspects of body movement5–8, but the mechanisms by which the SLR integrates internal/external stimulation and transmits locomotor commands to the spinal cord remain unclear. The lateral hypothalamic area (LHA) within the SLR has been implicated in motor control, particularly in motivated locomotion—internal need-driven movements such as food-seeking, exploration, or goal-directed behaviors9,10. However, the precise LHA circuits that control locomotion, particularly in the context of motivation, remain incompletely understood3,11. Recent single-neuron projectome studies have begun to reveal more detailed connections between specific neuronal subtypes in distinct brain regions, and these studies have provided insights into how these circuits may coordinate complex behaviors12–16.

Defining the neural circuits that control locomotion is also crucial for developing neural circuit-based strategies to promote motor recovery following injuries of the central nervous system (CNS). This is particularly relevant in the case of SCI, in which the brain loses its direct connection with the SC. Despite the neuronal death and axon degeneration that occur after SCI, some axons and dormant relay pathways often remain spared across the lesion site, providing a neural substrate that can be reactivated to promote functional recovery, even with limited axon regeneration17. Moreover, the restoration of locomotor function after SCI involves the reorganization of both spinal and supraspinal circuits18. Recent studies have revealed the contributions of specific brain regions to the recovery of motor function after SCI, further suggesting the importance of reactivating the neural circuits that are involved in regulating locomotion control19–22. However, effective therapeutic strategies for restoring motor function after SCI remain limited. As a promising strategy for in vivo neuronal activation, deep brain stimulation (DBS) offers the advantage of locally activating specific brain nuclei. Notably, Masini et al. successfully employed this approach to rescue motor deficits through the targeted manipulation of PPN glutamatergic neurons in a Parkinsonian mouse model23. Additionally, recent studies have revealed that targeting specific MLR regions, including cuneiform nucleus (CnF)24–26 and pedunculopontine nucleus (PPN)27, can enhance motor function and promote recovery in animal SCI models. However, the precise brain nuclei that should be targeted to achieve optimal motor recovery remain under these investigation28, and clinical trials are still in their early stages29,30.

Here, we employed a range of advanced techniques, including whole-brain trans-synaptic labeling, single-neuron projectome analysis, and projection-specific targeting, to investigate the contributions of the hypothalamic LHA nucleus in locomotion control and in motor function restoration following SCI. Our findings suggest that LHA facilitates motivated locomotion through an indirect pathway, particularly the pontine reticulospinal tract, rather than through direct projections to the spinal cord or through connections to the medial septum and diagonal band (MSDB). This indirect modulation of spinal circuits offers a target for therapeutic intervention. Moreover, we investigated the potential of DBS targeting the LHA to promote motor recovery, with the ultimate goal of developing a translatable strategy for restoring locomotion in individuals with severe SCI.

Results
LHA glutamatergic neurons are involved in hindlimb motor control
To visualize the hypothalamic areas that directly or indirectly project to the hindlimb muscles, we first injected pseudorabies virus (PRV), which encoded EGFP, directly into the tibialis anterior/gastrocnemius (TA/GS) muscle. This allowed for the retrograde trans-synaptic labeling of upstream neurons. The ClearMap31,32 method was utilized for automatic analysis and registration of volumetric data from cleared tissues 5.5 days after viral injection (see STAR Method). The reconstructed whole-brain data of labelled neurons were registered onto the standard Allen CCFv3 map, which showed a widespread distribution of PRV-labeled neurons across the brain (Fig. 1a and Supplementary Video 1). Further analysis revealed that labeled neurons were widely distributed across the hypothalamus, with the highest density of these neurons (36.6% of the total) in the LHA (Fig. 1b–d and Supplementary Fig. 1a, b).

Further kernel density estimation (KDE) of trans-synaptically retrogradely labeled neurons indicated that the highest density of labeled neurons was located in the caudal region of LHA (cLHA, Fig. 1e), which contains both excitatory and inhibitory neurons33. Further experiments on optogenetic stimulation of Vglut2-ChR2 expressing neurons in 10 hypothalamic nuclei in free-moving mice (Supplementary Fig. 1c–e) revealed that only the activation of LHA Vglut2+ neurons resulted in robust locomotion of the mice, in a frequency- and light intensity-dependent manner (Fig. 1f–h) (Ligh intensity: One-way ANOVA F(5, 30)=77.32 with p<0.0001, followed by Dunnett’s multiple comparison to 0.1 mW, vs 1 mW p=0.0034, and different from all other groups with p<0.0001. Frequency: One-way ANOVA F(4, 25)=19.11 with p<0.0001, followed by Dunnett’s multiple comparison to 0.1 Hz, different from all other groups with p<0.0001.) Thus, LHA Vglut2+ neurons represent the main population of hypothalamic neurons that are involved in regulating locomotion in mice.

LHA glutamatergic neurons regulate motivation-driven locomotion
To further understand how the firing rate of LHA neurons is related to locomotion speed, we performed single-unit recording at the caudal LHA of C57BL/6 mice walking on a linear track (1.5 m × 15 cm × 20 cm) using an 8-tetrode drivable array. A total of 156 neurons were recorded from 7 mice. Figure 1i shows an example neuron whose firing rate was positively correlated with speed. To minimize resting-state contamination, we applied a speed filter (excluding epochs below 5 cm/s)34 and found that 37 neurons (23.7% of the recorded cells) exhibited firing rates that were significantly modulated by locomotion speed (Spearman’s correlation, p<0.01; median p=0.42) (Fig. 1j). We subsequently applied two widely recognized criteria to categorize these neurons into putative principal cells, inferred to be glutamatergic neurons, and putative interneurons, believed to be GABAergic neurons35–37. Out of these 37 neurons, 24 neurons exhibited characteristics of putative glutamatergic neurons, 3 neurons exhibited characteristics of GABAergic neurons, and the remaining 10 neurons were indistinguishable (Supplementary Fig. 1f). Moreover, a subpopulation of neurons was found to exhibit an increased firing rate prior to the onset of locomotion (Fig. 1k). The LHA is known to be critical for regulating various locomotion-dependent physiological and behavioral functions, including predatory attack, evasion, and other motivated behaviors9,38,39. This functional diversity may arise from the heterogeneous cell populations and complex cytoarchitecture within the region. To investigate the association of excitatory versus inhibitory LHA neurons with motivation, we applied fiber photometry to record the Ca2+ signals of LHA neurons during food seeking. We injected AAV-DIO-GCaMP6s into the LHA of Vglut2-Cre or Vgat-Cre mice, allowing for the expression of GCaMP6s specifically in either excitatory or inhibitory LHA neurons, thereby facilitating fiber photometry imaging. We observed an increase of Ca2+ signals of excitatory neurons that was time locked to the onset of locomotion, with elevated activity during food seeking behavior when approaching the food. However, these signals decreased during the consumption period (Fig. 1l, m) (From resting to locomotion: p=0.0002, two-tailed, t-test with Welch’s correction, t=7.064, df=6.746. From food seeking to consumption: p<0.0001, two-tailed, t-test, t=6.080, df=12.). LHA inhibitory neurons, on the other hand, did not respond to the onset of locomotion and food-seeking behavior; but their Ca2+ activity increased during food consumption (Supplementary Fig. 1g, h) (From resting to locomotion: p=0.6226, two-tailed, t-test, t=0.5052, df=12. From food seeking to consumption: p<0.0001, two-tailed, t-test, t=10.98, df=12.). Moreover, in open field test, optical stimulation of LHA Vgat+ neurons did not induce locomotion in freely-moving mice (Supplementary Fig. 1i) (Maximum speed: p=0.3904, two-tailed, t-test, t=0.8978, df = 10. Total distance: p=0.3985, two-tailed, t-test, t=0.8819, df=10.).

To further explore the role of LHA Vglut2+ neurons in basic locomotor function and motivated locomotion (e.g., food-seeking), we expressed the diphtheria toxin (DT) receptor (DTR) by infusing AAV-DIO-DTR-mCherry vectors into LHA of Vglut2+-Cre mice (Fig. 1n). DT administration induced neuronal ablation in DTR-expressing mice but not in mCherry expressing control mice (Supplementary Fig. 1j) (mCherry+ cell numbers/section: p<0.0001, two-tailed, t-test, t=11.31, df=10.). After ablation of LHA excitatory neurons, no significant changes were observed in basic locomotor functions such as hindlimb protraction, retraction and maximum iliac crest height (Fig. 1o, p), suggesting that the ablation of LHA glutamatergic neurons did not impact basic locomotion (Hindlimb retraction: p=0.3885, two-tailed, t-test, t=0.9016, df=10. Hindlimb protraction: p=0.5318, two-tailed, t-test, t=0.6477, df=10. Maximum iliac crest height: p=0.8871, two-tailed, t-test, t=0.1456, df=10.). The ablation of LHA excitatory neurons resulted in no significant change in overall activity in an open field setting (Fig. 1q) (Center time: p=0.8479, two-tailed, t-test, t=0.1969, df=10. Total distance: p=0.4368, two-tailed, t-test, t=0.8100, df=10. Entries to center area: p=0.5639, two-tailed, t-test, t=0.5968, df=10.) However, in the open field chamber in which food pellets were placed in the center, the locomotor activity was reduced and the mice were less likely to enter the center area, even though their appetite, as measured by food pellet consumption, was not affected (Fig. 1r) (Entries to center area: p=0.0332, two-tailed, t-test, t=2.468, df=10. Total distance: p=0.0281, two-tailed, t-test, t=2.565, df=10. Number of pellet eaten: p=0.7288, two-tailed, t-test, t=0.3566, df=10.). Together, these results suggest that LHA neurons play a vital role in regulating motivational locomotion.

LHA neurons regulate locomotion via projections to PnO and MSDB
We next explored the circuit mechanism underlying the function of LHA projection in locomotion control. We searched in a single-neuron projectome dataset for mouse LHA for the major brain regions receiving axon projections from LHA neurons40. This dataset consists of single-axon tracing of 916 sparsely labelled neurons expressing various neuropeptides (Supplementary Fig. 2a). To classify the pattern of axon projections of these neurons, we calculated similarity scores based on the shortest distances between neuron pairs. We then performed hierarchical clustering of the similarity matrix of all neuron pairs using Ward’s linkage. Using this approach, we categorized lateral hypothalamus neurons into 4 main clusters and 7 projectome-defined subtypes (Fig. 2a, b and Supplementary Fig. 2b), with subtypes 1 and 2 in the rostral projecting cluster (Cluster 1, ~16% of the total neurons), subtype 3 in the local projecting cluster (Cluster 2, ~13% of the total neurons), subtypes 4 and 5 in the caudal projecting cluster (Cluster 3, ~46% of the total neurons), and subtypes 6 and 7 in the long-distance projecting cluster (Cluster 4, ~25% of the total neurons).

Among the neurons that were grouped into subtypes 1 and 2 of the rostral projecting cluster (Cluster 1), most neurons displayed targeting preferences for thalamus (TH) and cerebral nuclei (CNU), respectively. Notably, a distinct subgroup within Subtype 1 (TH-projecting) specifically projected to the lateral habenula (LHb). Subtype 2 exhibited strong projections to the cerebral nuclei (CNU), including lateral septal nucleus (LS), bed nuclei of the stria terminalis (BST), substantia innominate (SI), and medial septum and diagonal band of Broca (MSDB). Cluster 2 consisted of subtype 3, which primarily projected within the hypothalamus with short axons. By comparison, the midbrain-projecting subtype 4 in cluster 3 projected with varying strengths to the periaqueductal gray (PAG) and superior colliculus (SCm) in the midbrain. Neurons in subtype 5 within cluster 3 primarily terminated their axons within the pontine, including PnO, parabrachial nucleus (PB), laterodorsal tegmental nucleus (LDT), and pontine central gray (PCG). Cluster 4 neurons had broader projections over longer distances, with subtype 6 projecting specifically to the cortex and the medulla, while subtype 7 was more medulla-specific. Among cluster 4 neurons, nearly half (111 out of 233) of the neurons had direct projections to the spinal cord (Supplementary Fig. 2b).

Given that optogenetic activation of LHA glutamatergic neurons elicited hindlimb locomotion, we examined whether those neurons that projected directly to the lumbar spinal cord circuits (Fig. 2c) might generate rhythmic motor patterns. The results from anterograde tracing demonstrated that LHA glutamatergic neurons projected robustly to the dorsal part of lumbar spinal cord (Fig. 2d, e). Retrograde labeling also demonstrated the existence of spinal-projecting LHA glutamatergic neurons (Fig. 2f). To explore the function of the LHA-spinal cord pathway, we expressed ChR2 in excitatory spinal cord-projecting LHA neurons by injecting retrogradely transported AAV-expressing Cre-dependent ChR2 into the spinal cord and implanting optical fibers in the LHA of Vglut2-Cre mice. We found that optogenetic stimulation of these spinal cord-projecting LHA neurons did not elicit locomotor behaviors (Fig. 2i–k). This indicates that an indirect pathway from LHA to spinal cord might be involved.

On the basis of our hierarchical clustering analysis, anterograde tracing results and previous research in motor control1,4–6,41,42, we selected 3 potential downstream brain regions that receive projections from the rostral LHA neurons, including the LS, MSDB, and BST, as well as 7 downstream brain regions that receive projections from the caudal LHA neurons, including the substantia nigra reticular part (SNr), PB, LDT, PnO, PCG, gigantocellular reticular nucleus (Gi), and lateral paragigantocellular nucleus (LPGi), to determine their roles in locomotion (Fig. 2g, h and Supplementary Fig. 2c). This was achieved by injecting AAVs carrying Cre-dependent ChR2-mCherry into the LHA of Vglut2-Cre mice and stimulating the axonal terminals of LHA neurons in these downstream brain regions across the brain (Fig. 2i). Optogenetic activation of axon terminals of LHA neurons in these different regions resulted in varying effects on the mouse locomotion. Notably, activation of LHA neuron projection to the PnO or MSDB could initiate locomotion, whereas MSDB activation led to high-speed locomotion, in contrast to the more moderate responses elicited by PnO activation (Fig. 2j, k and Supplementary Video 2 and 3) (Total distance: One-way ANOVA F(11, 60)=265.0 with p<0.0001, followed by Dunnett’s multiple comparison to SC, different from LHA soma, MSDB and PnO with p<0.0001. Maximum speed: One-way ANOVA F(11, 60)=151.2 with p<0.0001, followed by Dunnett’s multiple comparison to SC, different from LHA soma, MSDB and PnO with p<0.0001.).

To further validate the involvement of the LHA-PnO and LHA-MSDB projections in locomotion, we injected retrogradely transported AAVs carrying Cre into the PnO and MSDB, respectively, while expressing Cre-dependent ChR2 in the LHA of wild-type mice. Stimulation of the cell bodies of LHA neurons projecting to PnO or MSDB successfully induced locomotion in freely moving mice (Supplementary Fig. 2d–h) (LHA-MSDB maximum speed: p<0.0001, two-tailed with Welch’s correction, t-test, t=26.32, df= 5.721. LHA-MSDB total distance: p<0.0001, two-tailed with Welch’s correction, t-test, t=22.47, df=5.121. LHA-PnO maximum speed: p<0.0001, two-tailed with Welch’s correction, t-test, t=14.70, df=5.273. LHA-PnO total distance: p<0.0001, two-tailed, t-test, t=15.37, df=10.). To further confirm the functional role of LHA glutamatergic projections to the MSDB/PnO, we employed an intersectional optogenetic strategy in Vglut2-Cre mice by injecting RetroAAV-DIO-FLP into the PnO or MSDB and AAV-fDIO-ChR2 into the LHA, followed by optical stimulation in the LHA. Importantly, this approach robustly induced locomotion, which was consistent with our original findings in WT mice (Fig. 2l–n and Supplementary Fig. 2i, j) (LHA-MSDB maximum speed: p<0.0001, two-tailed with Welch’s correction, t-test, t=17.57, df=5.479. LHA-MSDB total distance: p<0.0001, two-tailed with Welch’s correction, t-test, t=17.90, df=5.704. LHA-PnO maximum speed: p<0.0001, two-tailed with Welch’s correction, t-test, t=10.30, df=5.880. LHA-PnO total distance: p=0.0003, two-tailed with Welch’s correction, t-test, t=8.279, df=5.444.).

Given that low speed locomotion is controlled by the caudal PPN5,23,34,43, we tested the potential role of PPN glutamatergic neurons in mediating LHA-PnO induced locomotion. The PPN glutamatergic neurons were selectively silenced by bilaterally injecting AAV-DIO-hM4Di into the caudal PPN of Vglut2-Cre mice and administering CNO before locomotor testing (Supplementary Fig. 2k, l). LHA-PnO optogenetic stimulation still evoked locomotion even after PPN glutamatergic neurons inhibition (Supplementary Fig. 2m, n) (Maximum speed: p=0.6738, two-tailed, t-test, t=0.4335, df=10. Total distance: p=0.7968, two-tailed, t-test, t=0.2645, df=10.). These results demonstrate that LHA-PnO projections can drive locomotion independently of PPN glutamatergic neurons, further supporting our conclusion that the LHA-PnO pathway is crucial for locomotion initiation. Collectively, these results demonstrate that the projections from the LHA to the PnO or MSDB play a crucial role in regulating locomotion.

LHA-PnO neurons facilitate motivation-driven locomotion without inducing anxiety
Next, we investigated the differential roles of MSDB-projecting and PnO-projecting LHA neurons in regulating mouse locomotion. Our findings demonstrated that optical stimulation of MSDB-projecting LHA neurons elicited high-speed running that resembled escape behavior, a response distinct from that observed following stimulation of LHA-PnO projections (Fig. 2j, k). Further open field test and elevated plus-maze test for the stress response of the mice indicated that stimulation of MSDB-projecting LHA neurons resulted in a significant reduction in the time spent in the center during the open field test and in the time in the open arms during the elevated plus-maze test. In contrast, stimulation of LHA-PnO axon terminals did not elicit any significant stress response (Fig. 3a–c) (LHA-MSDB OFT center time: One-way ANOVA F(2, 15)=21.50 with p<0.0001, followed by Tukey’s multiple comparison, before vs during stim p<0.0001, before vs after stim p=0.0004, and during vs after stim p=0.5784. LHA-MSDB OFT total distance: One-way ANOVA F(2, 15)=23.19 with p<0.0001, followed by Tukey’s multiple comparison, before vs during stim p=0.002, before vs after stim p=0.0572, and during vs after stim p<0.0001. LHA-MSDB OFT entries to center area: One-way ANOVA F(2, 15)=14.42 with p=0.0003, followed by Tukey’s multiple comparison, before vs during stim p=0.0007, before vs after stim p=0.001, and during vs after stim p=0.9794. LHA-PnO OFT center time: One-way ANOVA F(2, 15)=1.512 with p=0.2522. LHA-PnO OFT total distance: One-way ANOVA F(2, 15)=4.466 with p=0.0301, followed by Tukey’s multiple comparison, before vs during stim p=0.0236, before vs after stim p=0.3771, and during vs after stim p=0.2725. LHA-PnO entries to center area: One-way ANOVA F(2, 15)=20.57 with p<0.0001, followed by Tukey’s multiple comparison, before vs during stim p<0.0001, before vs after stim p=0.006, and during vs after stim p=0.4792. LHA-MSDB EPM center time: One-way ANOVA F(2, 15)=40.55 with p<0.0001, followed by Tukey’s multiple comparison, before vs during stim p<0.0001, before vs after stim p<0.0001, and during vs after stim p=0.5430. LHA-MSDB EPM total distance: One-way ANOVA F(2, 15)=10.02 with p=0.0017, followed by Tukey’s multiple comparison, before vs during stim p=0.0433, before vs after stim p=0.2118, and during vs after stim p=0.0013. LHA-PnO EPM center time: One-way ANOVA F(2, 15)=1.629 with p=0.2290. LHA-PnO EPM total distance: One-way ANOVA F(2, 15)=3.640 with p=0.0515, followed by Tukey’s multiple comparison, before vs during stim p=0.0413, before vs after stim p=0.4069, and during vs after stim p=0.3761. Notably, during the food-seeking test, optogenetic activation of the LHA-PnO pathway increased the number of entries into the center zone without affecting food consumption, whereas stimulation of LHA-MSDB terminals produced the opposite behavioral effect, significantly reducing center entries (Supplementary Fig. 3b, c) (LHA-MSDB entries to center area: p=0.0028, two-tailed, t-test, t=3.931, df=10. LHA-MSDB total distance: p=0.2008, two-tailed, t-test, t=1.369, df=10. LHA-MSDB pellet eaten: p=0.8376, two-tailed, t-test, t=0.2104, df=10. LHA-PnO entries to center area: p=0.0252, two-tailed, t-test, t=2.629, df=10. LHA-PnO total distance: p=0.2954, two-tailed, t-test, t=1.104, df=10. LHA-PnO pellet eaten: p=0.5732, two-tailed, t-test, t=0.5824, df=10.). Through injecting retrogradely transported AAVs carrying Cre into the PnO or the MSDB, while expressing Cre-dependent DTR in the LHA of wild-type mice, we found that depletion of PnO-projecting rather than MSDB-projecting LHA neuron impaired motivation-driven locomotion, as indicated by the reduced total distance and entries to the center compared to the control group (Fig. 3d, e) (LHA-MSDB entries to center area: p=0.4456, two-tailed, t-test, t=0.7941, df=10. LHA-MSDB total distance: p=0.4755, two-tailed, t-test, t=0.7414, df=10. LHA-MSDB pellet eaten: p=0.7094, two-tailed, t-test, t=0.3835, df=10. LHA-PnO entries to center area: p=0.0051, two-tailed, t-test, t=3.571, df=10. LHA-PnO total distance: p=0.0293, two-tailed, t-test, t=2.541, df=10. LHA-PnO pellet eaten: p=0.3122, two-tailed, t-test, t=1.064, df=10.). However, ablation of MSDB- or PnO-projecting LHA neurons did not significantly alter general locomotor activity in the open field test nor anxiety-like behavior in the elevated plus-maze (Supplementary Fig. 3d–f) (LHA-MSDB OFT center time: p=0.9432, two-tailed, t-test, t=0.07311, df=10. total distance: p=0.9087, two-tailed, t-test, t=0.1176, df=10. Entries to center area: p=0.7666, two-tailed, t-test, t=0.3050, df=10. LHA-MSDB EPM open arm time: p=0.6752, two-tailed, t-test, t=0.4316, df=10. total distance: p=0.9433, two-tailed, t-test, t=0.07293, df=10. LHA-PnO OFT center time: p=0.8747, two-tailed, t-test, t=0.1618, df=10. total distance: p=0.8570, two-tailed, t-test, t=0.1848, df=10. Entries to center area: p=0.9585, two-tailed, t-test, t=0.05341, df=10. LHA-MSDB EPM open arm time: p=0.7860, two-tailed, t-test, t=0.2789, df=10. total distance: p=0.8669, two-tailed, t-test, t=0.1720, df=10.). Immunohistochemical analysis confirmed efficiency of ablation of LHA-PnO projecting neurons (Supplementary Fig. 3g). Collectively, the data demonstrated that while LHA neurons are dispensable for baseline locomotor activity, the LHA-PnO pathway was specifically required for motivation-driven locomotion during food-seeking behavior. These findings establish the LHA-PnO circuit as a dedicated neural substrate that selectively transforms motivational signals into locomotor output, without affecting general movement. Further examination of the soma distribution of neurons projecting to these two brain areas in single-neuron projectome data showed that somata for these two distinct projections exhibited a spatially distinct anterior-posterior arrangement (Fig. 3f and Supplementary Fig. 3h). According to the connectome data, different projecting clusters exhibited distinct peptidergic signatures (Supplementary Fig. 3i). Approximately 48.99% of neurons that project to the spinal cord express Orexin, while neurons projecting to the PnO and MSDB comprise a heterogeneous population of peptidergic neurons. Axonal collaterals play a crucial role in neuronal communication, allowing for the integration of signals across different regions. Topographic arrangements of individual PnO-projecting neuron revealed the existence of axon collaterals in both PPN and VTA (Ventral tegmental area), which have been reported to play roles in locomotion and motivational circuits, respectively (Supplementary Fig. 3j)5,6,9. A strategy for intersectional viral screening was also performed to identify collateral innervation of PnO-projecting LHA neurons. RetroAAV-Cre was injected in PnO, followed by injection of AAV-DIO-mChery in LHA. Labeling of PnO-projecting LHA neurons revealed collateral axonal processes in the VTA and PPN (Supplementary Fig. 3k). These findings indicate a complex interplay between axonal projection patterns, somatic locations, and neuropeptide expression, which are intrinsic features of specific neuronal populations. Therefore, integrating projectome-defined subtypes with molecular markers and somatic locations is expected to enhance our understanding of the distinct neuronal subtypes that are involved in diverse hypothalamic functions.

Activation of the LHA-PnO-Spinal pathway initiates locomotion
Our anterograde tracing experiment showed that PnO comprised a majority of pontine reticulospinal neurons which directly projected to the spinal cord (Fig. 3g, h). Through retrogradely labeling from the lumbar spinal cord, we found that somata of spinal-projecting PnO excitatory neurons were mainly located in the lateral part of PnO (Fig. 3i, j). To demonstrate the role of PnO as a relay station connecting LHA and lumbar spinal cord, AAVretro-DIO-EYFP was injected into lumbar spinal cord, followed by an anterograde tracing virus AAV-DIO-mCherry injected into LHA. Double positive signals were identified in PnO (Supplementary Fig. 3l). Moreover, we performed rabies virus-based three-step monosynaptic retrograde tracing experiments. Specifically, retroAAV-hSyn-Cre was delivered into the lumbar spinal cord, followed by the injection of AAVs expressing Cre-dependent avian-specific retroviral receptor (TVA) and rabies virus glycoprotein (RVG) into the PnO 2 weeks later. RV-EnvA-dsRed was then delivered into PnO, and neurons expressing both EGFP and dsRed in PnO represented the “starter cells” in the monosynaptic rabies virus tracing method. Furthermore, trans-synaptically labelled neurons were abundantly found in the LHA (Fig. 3k). These results indicate the existence of LHA-PnO-spinal cord pathway.

We then expressed anterograde trans-synaptic AAVs (AAV serotype 1) carrying Cre into the LHA in WT mice44–46, alongside AAV encoding Cre-dependent ChR2 in the PnO to stimulate the LHA-recipient PnO neurons. Optogenetic activation of LHA-recipient PnO neurons produced locomotor effects that were similar to those observed when stimulating LHA glutamatergic terminals in the PnO, confirming that the LHA-PnO pathway is crucial for locomotion (Fig. 3l and Supplementary Video 4) (Maximum speed: p<0.0001, two-tailed, t-test, t=20.18, df=10. Total distance: p<0.0001, two-tailed, t-test, t=30.72, df=10.). To functionally investigate whether the PnO-spinal pathway plays a direct role in locomotor control, we delivered retroAAV-DIO-ChR2 into the spinal cord of Vglut2-Cre mice. This retrograde-transported viral construct was specifically taken up by axon terminals of spinally-projecting neurons and transported in a retrograde manner to their cell bodies in the PnO. Following ChR2 expression in retrogradely labeled cell bodies in the PnO, optogenetic stimulation reliably induced locomotion in freely-moving mice (Supplementary Fig. 3m) (Maximum speed: p<0.0001, two-tailed, t-test, t=13.82, df=10. Total distance: p<0.0001, two-tailed, t-test, t=19.43, df=10.). To further validate the role of LHA-PnO-Spinal cord, we conducted optogenetic experiments in Vglut2-cre mice. Specifically, we injected AAV1-FLP into LHA and Retro-ConFon-ChR2 into the spinal cord. Optical stimulation of PnO neurons that both received LHA inputs and projected to the spinal cord evoked locomotion (Fig. 3m) (Maximum speed: p<0.0001, two-tailed, t-test, t=10.02, df=10. Total distance: p<0.0001, two-tailed, t-test, t=12.35, df=10.). Together, these results indicate that LHA indirectly activates spinal cord neurons through a relay station of PnO to induce locomotion behavior.

LHA-PnO circuit contributes to spontaneous locomotor recovery following incomplete SCI
Our results demonstrated that the LHA-PnO-Spinal descending pathway plays a crucial role in facilitating motivated locomotion in uninjured mice. This prompted us to explore their potential involvement in voluntary locomotion following SCI. We first characterized spontaneous locomotor recovery in mice received a lateral hemisection at the thoracic segment 10 (T10)21,47. Three days after injury, mice displayed a complete loss of stepping ability in the ipsilesional hindlimb. In contrast, in the chronic phase after SCI, we observed significant locomotor recovery in all mice, as indicated by body weight-bearing plantar stepping (Supplementary Fig. 4a, b).

Next, we examined whether LHA neurons contribute to the observed locomotor recovery after spinal hemisection by performing two complementary sets of experiments (Fig. 4a, b). Three groups of WT mice received PRV-injection from the hindlimb (GS/TA) muscle and sacrificed 5.5 days after injection: uninjured control mice, mice injected 3 days after injury, and mice injected 8 weeks after injury. In mice that were injected with PRV 3 days post-injury, we found that neuronal labeling in LHA was much lower than that observed in uninjured mice, indicating spinal hemisection disrupted descending projection from LHA to the locomotor circuits below the lesion. In contrast, mice injected with the PRV 8 weeks post-injury demonstrated a significant recovery of labeled neurons in cLHA, but not in rostral LHA (rLHA), suggesting substantial re-establishment of neuronal connections from cLHA to the spinal cord over time after SCI (Fig. 4b and Supplementary Fig. 4c) (One-way ANOVA F(2, 6)=7.576 with p=0.0228, followed by Tukey’s multiple comparison, intact vs acute p=0.0273, intact vs chronic p=0.8972, and acute vs chronic p=0.0466. Additionally, we mapped the LHA regions responsible for triggering TA muscle contraction in uninjured mice and injured mice during the early and late post-injury phase under anesthesia. In uninjured mice, electrical stimulation of the LHA induced TA muscle contraction. Stimulation at the same LHA subregion failed to produce TA muscle contraction at the early stage (1 week post-injury), but successfully triggered TA muscle activation 8 weeks post-injury (Fig. 4b). This suggests a re-establishment of functional connections between LHA and the spinal cord over time following SCI.

Given the re-establishment of LHA-spinal cord connections during spontaneous recovery after incomplete SCI, we further evaluated whether LHA glutamatergic neurons are necessary for the functional restoration of locomotion. To selectively ablate LHA glutamatergic neurons, we bilaterally injected AAV-DIO-DTR into the LHA of Vglut2-Cre mice, followed by DT administration to induce targeted neuronal ablation. After conditional genetic ablation of LHA glutamatergic neurons in hemisection injured mice, we measured basic over-ground locomotion and motivational vigor in fasting-triggered food-seeking behaviors (Fig. 4c). We found that mice with viral-induced depletion of LHA Vglut2+ neurons exhibited impaired recovery of locomotor function, as characterized by pronounced paw dragging and impaired protraction of the denervated hindlimb (Fig. 4d–f) (Hindlimb retraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p<0.0001, 2w p<0.0001, 4w p=0.0006. Hindlimb protraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p>0.9999, 2w p<0.0001, 4w p=0.0483. Height of iliac crest: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p=0.8425, 2w p>0.9999, 4w p>0.9999. Although locomotor function was impaired, general locomotor activity did not significantly differ from that of the controls (Supplementary Fig. 4d) (Center time: p=0.6265, two-tailed, t-test, t=0.5020, df=10. Total distance: p=0.4188, two-tailed, t-test, t=0.8432, df=10. Entries to center area: p=0.8890, two-tailed, t-test, t=0.1431, df=10.). However, in the food seeking test, we observed a significant reduction in both total distance and center entries, indicating the deficit in motivation-driven locomotion rather than generalized motor activity (Fig. 4g and Supplementary Fig. 4e) (1 week after injury: Entries to center area: p<0.0001, two-tailed, t-test, t=7.447, df=10. Total distance: p=0.0003, two-tailed, t-test, t=5.453, df=10. Number of pellet eaten: p=0.5971, two-tailed, t-test, t=0.5459, df=10. 2 weeks after injury: Entries to center area: p<0.0001, two-tailed, t-test, t=6.936, df=10. Total distance: p=0.0009, two-tailed, t-test, t=4.681, df=10. Number of pellet eaten: p=0.7914, two-tailed, t-test, t=0.2717, df=10.). To specifically deplete PnO-projecting LHA neurons, we injected retrogradely transported AAVs carrying Cre into the PnO while expressing Cre-dependent DTR in the LHA of wild-type mice. This genetic ablation of PnO-projecting LHA neurons also significantly impaired the functional recovery of SCI mice during over-ground locomotion, as evidenced by pronounced paw dragging and impaired protraction of the denervated hindlimb (Fig. 4h–j) (Hindlimb retraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p<0.0001, 2w p<0.0001, 4w p=0.0406. Hindlimb protraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p>0.9999, 2w p<0.0001, 4w p=0.0163. Height of iliac crest: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p>0.9999, 2w p>0.9999, 4w p>0.9999. No significant difference in general locomotor activity was observed compared with that of controls (Supplementary Fig. 4f) (Center time: p=0.8937, two-tailed, t-test, t=0.1371, df=10. Total distance: p=0.7823, two-tailed, t-test, t=0.2838, df=10. Entries to center area: p=0.7089, two-tailed, t-test, t=0.3842, df=10.). Moreover, impaired functional recovery was associated with decreased food-seeking motivational locomotion after SCI (Fig. 4k and Supplementary Fig. 4g) (1 week after injury: Entries to center area: p<0.0001, two-tailed, t-test, t=6.713, df=10. Total distance: p=0.0006, two-tailed, t-test, t=4.877, df=10. Number of pellet eaten: p>0.9999, two-tailed, t-test, t=0.000, df=10. 2 weeks after injury: Entries to center area: p=0.0001, two-tailed, t-test, t=6.195, df=10. Total distance: p=0.0003, two-tailed, t-test, t=5.414, df=10. Number of pellet eaten: p=0.7137, two-tailed, t-test, t=0.3775, df=10.). These findings suggest that motivational locomotion encoded by PnO-projecting LHA neurons is important for natural repair after incomplete SCI.

Subsequently, we examined the post-injured period in which LHA neuronal activity could exert their action in promoting locomotor recovery. This was achieved by injecting AAVs carrying Cre-dependent ChR2-mCherry into LHA of Vglut2-Cre mice prior to SCI, and optogenetically stimulated LHA at 1, 4, and 8 weeks after injury (Fig. 4l). All tested SCI mice demonstrated significantly improved hindlimb locomotion during the period of light stimulation. Detailed kinematic analysis revealed increased weight support and enhanced hindlimb stepping ability (Fig. 4m, n and Supplementary Video 5) (Hindlimb retraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p<0.0001, 4w p<0.0001, 8w p=0.0006. Hindlimb protraction: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p<0.0001, 4w p<0.0404, 8w p=0.0383. Height of iliac crest: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p<0.0001, 4w p=0.0039, 8w p=0.0065. These results indicate that LHA neuronal activity results in immediate facilitation of hindlimb motor functions in hemisectioned SCI mice.

Acute LHA stimulation enables stepping in paralyzed mice after stagger SCI
We next investigated whether more clinically relevant DBS at the LHA could improve locomotion in mice with severe SCI. Results described above (Fig. 3b, c) indicated that stimulation of LHA-MSDB pathway induced significant anxiety-like behaviors that are known to be detrimental to recovery48,49. Therefore, we aimed to selectively stimulate the LHA-PnO pathway by adjusting the locus of stimulation within the LHA during DBS.

On the basis of the soma distribution of LHA neurons that project to the PnO and MSDB, we identified that the LHA neurons projecting to PnO and MSDB are primarily located in the caudal and rostral region of the LHA (Fig. 3f), respectively. We injected AAVs carrying GCaMP6s into both PnO and MSDB and recorded the activation of these downstream brain regions during electrical stimulation of various locations along the anterior-posterior axis of the LHA (Fig. 5a, b). In head-fixed mice, we found that stimulating the rostral (−1.0 mm from the bregma) and caudal (−1.5 mm from the bregma) LHA resulted in preferential activation of MSDB and PnO, respectively (Fig. 5c–f) (rLHA DBS: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, before stimulation p>0.9999, during stimulation p<0.0001. cLHA DBS: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, before stimulation p>0.9999, during stimulation p<0.0001. Moreover, electrical stimulation in caudal LHA also induced elevated locomotion immediately in free-moving uninjured mice (Fig. 5g–k) (Total distance: p<0.0001, two-tailed, t-test with Welch’s correction, t=14.04, df= 6.135. Maximum speed: p<0.0001, two-tailed with Welch’s correction, t-test, t=13.92, df=5.226.). These results prompted us to apply DBS at the specific location of LHA to facilitate functional recovery following severe SCI.

We established a staggered SCI model with opposite lateral hemisection at T7 and T10, creating a more challenging scenario by eliminating brain-derived innervation below T10 while maintaining a spared tissue bridge between the two lesions to relay descending signals17,50. The mice with this staggered lesion exhibited nearly complete and permanent hindlimb paralysis. The preserved intersegmental relay circuits between the lesions make this model suitable for studying neuromodulation-based recovery strategies. We conducted a time course experiment to assess the effects of LHA-DBS on locomotor function by evaluating animals with and without LHA-DBS at post-injury weeks 1, 4, and 10 and analyzing the immediate effect on hindlimb stepping (Fig. 5l, m). We observed no hindlimb responses to LHA-DBS at 1 week post-injury, but we observed significantly improved walking ability during DBS at 4 weeks post-injury, as indicated by significant increases in maximum iliac crest height and stride length. This DBS effect was observed continuously until at least 10 weeks post-injury (Fig. 5n–p) (Maximum crest height: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p=0.2015, 4w p=0.0250, 10w p=0.0013. Stride length: Two-way RM ANOVA followed by Bonferroni’s multiple comparison, 1w p=0.2666, 4w p=0.0005, 10w p<0.0001. 1 week vs 10 weeks p<0.0001 and 4 weeks vs 10 weeks p<0.0001. Inter-ventral cFos+: One-way ANOVA F(2, 12)=51.29 with p<0.0001, followed by Tukey’s multiple comparison, 1 week vs 4 weeks p=0.0033, 1 week vs 10 weeks p<0.0001, and 4 weeks vs 10 weeks p=0.0002. Notably, the behavioral improvement by DBS described above was not maintained after the cessation of stimulation. These findings led to our further investigation of the potential of repetitive long-term LHA-DBS for promoting functional locomotor recovery after SCI.

Intention-gated long-term LHA-DBS enhances locomotor recovery after sever SCI
Previous findings have demonstrated that the delivery of DBS triggers animal’s stress-like responses27,53. We also observed elevated stress-like response under the condition of LHA-DBS, even for localized caudal LHA stimulation. To reduce this side effect, we applied an intention-gated stimulation algorithm that uses animal’s locomotor intention signals to trigger LHA-DBS. We decoded the intention of locomotion with a 32-channel micro-wire array electrode that was implanted in the multi-unit activity (MUA) in the hindlimb region of the motor cortex (M1HL)27,54 and used the decoded signal to stimulate the LHA via a brain-computer interface (BCI)-DBS system. We found that locomotor intentional signals, as indicated by an increased MUA firing rate prior to the initiation of hindlimb movements, could be observed in both intact and staggered SCI mice (Fig. 6a, b). Furthermore, open-field tests showed that this intention-gated LHA-DBS did not provoke stress-like responses in either healthy or injured mice (Fig. 6c–f) (Intact center time: One-way ANOVA F(2, 15)=15.59 with p=0.0002, followed by Tukey’s multiple comparison, control vs DBS p=0.0033, control vs BCI-DBS p=0.3623, and DBS vs BCI-DBS p=0.0002. Intact total distance: One-way ANOVA F(2, 15)=8.137 with p=0.004, followed by Tukey’s multiple comparison, control vs DBS p=0.0445, control vs BCI-DBS p=0.0034, and DBS vs BCI-DBS p=0.4180. Intact entries to center area: One-way ANOVA F(2, 15)=23.80 with p<0.0001, followed by Tukey’s multiple comparison, control vs DBS p=0.0066, control vs BCI-DBS p=0.0134, and DBS vs BCI-DBS p<0.0001. Stagger injury center time: One-way ANOVA F(2, 15)=64.27 with p<0.0001, followed by Tukey’s multiple comparison, control vs DBS p=0.5107, control vs BCI-DBS p<0.0001, and DBS vs BCI-DBS p<0.0001. Stagger injury total distance: One-way ANOVA F(2, 15)=38.00 with p<0.0001, followed by Tukey’s multiple comparison, control vs DBS p<0.0001, control vs BCI-DBS p<0.0001, and DBS vs BCI-DBS p=0.3611. Stagger injury entries to center area: One-way ANOVA F(2, 15)=53.58 with p<0.0001, followed by Tukey’s multiple comparison, control vs DBS p=0.0022, control vs BCI-DBS p<0.0001, and DBS vs BCI-DBS p<0.0001.

The above findings suggest that an intention-gated BCI-DBS system could be used as a long-term treatment for promoting locomotor recovery after SCI. To test this hypothesis, we apply daily 1-h BCI-DBS treatment sessions to staggered SCI mice for 6 weeks, beginning at 4 weeks post-injury (Fig. 6g). The locomotor function of the mice was assayed by BMS scores weekly at the last day of the treatment without and in the present of DBS. In contrast to the un-treated SCI group, long-term BCI-DBS training resulted in significant functional recovery, including restored hindlimb stepping ability and increased BMS scores without DBS, although DBS during testing could further elevate the motor function (Fig. 6h, i and Supplementary Video 6). Kinematic analysis revealed substantial improvements in locomotion compared with that of the untreated group. For over-ground movement, the mice were able to support their body weight, showed significantly increased hindlimb oscillation, and exhibited muscle activity as confirmed by electromyogram (EMG) recordings (Fig. 6j, k) (Maximal crest height: One-way ANOVA F(2, 12)=48.43 with p<0.0001, followed by Tukey’s multiple comparison, control vs training without stim p=0.0017, control vs training with stim p<0.0001 and training without stim vs training with stim p=0.0005. Stride length: One-way ANOVA F(2, 12)=141.9 with p<0.0001, followed by Tukey’s multiple comparison, control vs training without stim p=0.0002, control vs training with stim p<0.0001, and training without stim vs training with stim p<0.0001. Furthermore, long-term BCI-DBS training led to a significant increase in the number of cFos-positive and cFos/Zfhx3 double-positive neurons in the inter-lesion spinal cord (Supplementary Fig. 6a–c) (cFos+ Zfhx3+/Zfhx3+: One-way ANOVA F(2, 12)=143.5 with p<0.0001, followed by Tukey’s multiple comparison, control vs training without stim p=0.0084, control vs training with stim p<0.0001 and training without stim vs training with stim p<0.0001. Inter-ventral cFos+: One-way ANOVA F(2, 12)=66.49 with p<0.0001, followed by Tukey’s multiple comparison, control vs training without stim p=0.0044, control vs training with stim p<0.0001, and training without stim vs training with stim p<0.0001. Collectively, these results indicate that long-term intention-gated LHA-DBS treatment effectively promotes locomotor recovery.

Methods
Mousestrains
The following mouse lines were used in this study: wild-type C57BL/6 (SLAC Laboratory Animal, Shanghai), Vglut2-ires-Cre mice (The Jackson Laboratory, Stock 016963), Vgat-ires-Cre mice (The Jackson Laboratory, Stock 016962). Eight- to twelve-week-old male and female mice were randomly assigned to experimental groups. All mice were housed under consistent conditions with unrestricted access to food and water and maintained on a 12-h light/dark cycle (light on from 7:00 a.m. to 7:00 p.m.). Before food seeking test, mice were fasted for 24 h. All animal studies and experimental procedures received approval from the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences.

Retrograde trans-synaptic PRV tracing
In the retrograde trans-synaptic tracing experiments, mice were anesthetized with intraperitoneal injection of Zoletil 50 (30 mg/kg) in combination with Xylazine Hydrochloride (40 mg/kg) and placed on the surgical table. An incision was made on the left to expose the tibialis anterior muscle. Three injection sites were made at a depth of 0.5–1.0 mm into the muscle through the microliter syringes (Hamilton). At each site, 1 μL PRG-EGFP (Braincase) was injected. Following the PRV injection, the incision was sutured. For histological analysis, mice were perfused at 5.5 days after the PRV injection.

Transparency and 3D image acquisition
All harvested samples were post-fixed overnight at 4 °C in 4% PFA in PBS. The solution of Benzyl Alcohol and Benzyl Benzoate (BABB) was then used to match the refractive index of the tissue. Cleared samples were imaged in a transverse orientation on a light-sheet microscope. The samples were scanned using the continuous light-sheet scanning method with the included contrast blending algorithm for the 564 nm channels. Cell detection was performed with the newly developed open-source ClearMap software73, tailored for cell nuclei, which used a background subtraction via morphological opening, followed by a sequence of filters, morphological operations, and a 3D peak detection. The color of the bubble corresponds to n2 fold change in cell density in HY.

Stereotactic surgery
Stereotactic injections into both the brain and spinal cord were performed using a microinjection syringe pump (World Precision Instruments, WPI) and a stereotactic apparatus (RWD Life Science). Prior to surgery, mice were anesthetized by intraperitoneal injection of Zoletil 50 (30 mg/kg) in combination with Xylazine Hydrochloride (40 mg/kg).

For the stereotactic injections into the brain, the skull of the head-immobilized mouse was exposed and treated with 3% hydrogen peroxide to remove connective tissue from the surface. The skull was then leveled anteroposteriorly between the bregma and lambda, and mediolaterally between the left and right hemispheres. A small hole was drilled in the skull at the precise coordinates corresponding to the target brain region. Virus was delivered to the target brain region at a rate of 50 nL/min using the microinjection syringe pump (WPI). When the injection was completed, the micropipette was held in place for 10 min, then raised up by 0.1 mm following an additional 3 min, and finally withdrawn at a low speed.

The coordinates for different brainstem targets are as follows (zero from Bregma; AP, anterior-posterior; ML, medial-lateral; DV, dorsal-ventral from dura; in mm): LHA, AP -1.58, ML ±1.0, DV 4.95; PnO, AP -4.3, ML ±0.85, DV 3.95; MSDB, AP +1.00, ML ±0.10, DV 3.60; M1, AP -0.7 to -1.2, ML ±1.0, DV 0.55; Gi, AP -6.0, ML ±0.35, DV 5.10; SNr, AP -3.2, ML ±1.15, DV 4.30; LPGi, AP -6.90, ML ±1.10, DV 5.00; MLR, AP -4.80, ML ±1.19, DV 2.70; LDT, AP -5.20, ML ±0.70, DV 2.50; PCG, AP -5.50, ML ±0.40, DV 3.1; LHA rostral part, AP -1.06, ML ±1.0, DV 4.95; LHA caudal part, AP -2.00, ML ±1.0, DV 4.95.

To implant an optical fiber in the target brain region for optical stimulation or fiber photometry recording, we drilled 3 to 4 small holes into the surface of the skull and inserted skull screws. Simultaneously, we polished the surface of skull with a cranial drill to increase friction.

After drilling the hole at the coordinates corresponding to the target brain region, the optical fiber was slowly lowered into the brain using the stereotactic arm at a speed of 1 mm/min. Once the optical fiber was in place, the gap in the skull was first filled with a biocompatible adhesive, and this was followed by primary fixation with light-curing adhesive, and finally the optical fiber was secured to the skull with dental cement.

To retrogradely target supraspinal neurons, we injected viruses into the lumbar segment of spinal cord. For the stereotactic injection into the spinal cord, a dorsal incision was made along the spine at the level of the lumbar segment. The overlying muscles were stripped to expose the lumbar spine, and a laminectomy was performed to expose the spinal cord. The virus was injected into the lumbar spinal cord at a rate of 60 nL/min. After the injection, the micropipette was held in place for a few minutes before it was slowly withdrawn.

Viral vectors in experiments
For optogenetic activation of LHA in Figs. 1 and 4 and Supplementary Figs. 1 and 4, AAV2/9-EF1α-DIO-ChR2-mCherry (BrianVTA; 5.6×1012) was injected into the LHA. For fiber photometry experiments in Figs. 1, 5 and Supplementary Fig. 1, AAV2/9-hSyn-DIO-GCaMP6s (BrianCase; 6.7×1012) was injected into the LHA. For deletion of LHA in Figs. 1 and 4, AAV2/9-EF1α-DIO-DTR-mCherry (BrianVTA; 6.0 ×1012) was injected into the LHA. For retrograde anatomical tracing experiments in Figs. 2 and 3, AAV2/2Retro-EF1α-DIO-ChR2-EYFP (BrianCase; 5.7×1012) was injected into the spinal cord. For optogenetic activation of LHA-PnO or MSDB pathway in Vglut2-Cre mice in Fig. 2, AAV2/2Retro-EF1α-DIO-FLP (BrianVTA; 4.7×1012) was injected into PnO or MSDB, and AAV2/9-hSyn-fDIO-ChR2-mCherry (BrianVTA; 5.0×1012) was injected into the LHA. For optogenetic activation of LHA-PnO or MSDB pathway in WT mice in Supplementary Fig. 2, AAV2/2Retro-EF1α-Cre (BrianVTA; 5.4×1012) was injected into PnO or MSDB, and AAV2/9-EF1α-DIO-ChR2-mCherry (BrianVTA; 5.6×1012) was injected into the LHA. For chemogenetic inhibition of the PPN in Supplementary Fig. 2, AAV2/9-EF1α-DIO-hM4Di-EGFP (BrianCase; 6.4×1012) was bilaterally injected into the PPN. For deletion of LHA-PnO or MSDB pathway in Fig. 3, AAV2/2Retro-EF1α-Cre (BrianVTA; 5.4×1012) was injected into PnO or MSDB, AAV2/9-EF1α-DIO-DTR-mCherry (BrianVTA; 6.0×1012) was injected into the LHA. For anterograde anatomical tracing experiments in Fig. 3, AAV2/9-EF1α-DIO-ChR2-mCherry (BrianVTA; 5.6×1012) was injected into the PnO. For optogenetic activation of PnO neurons receiving LHA projection in Fig. 3, AAV2/1-hSyn-Cre (BrianVTA; 1.4×1013) was injected into the LHA, and AAV2/9-EF1α-DIO-ChR2-mCherry (BrianVTA; 5.6×1012) was injected into the PnO. For optogenetic activation of PnO-spinal cord in Supplementary Fig. 3, AAV2/2Retro-EF1α-DIO-ChR2-EYFP (BrianCase; 4.2×1012) was injected into the spinal cord. For anatomical tracing of LHA-PnO-spinal cord pathway in Fig. 3, AAV2/2Retro-EF1α-Cre (BrianVTA; 5.4×1012) was injected into the spinal cord, AAV2/8-EF1α-DIO-H2B-EGFP-T2A-TVA (BrianVTA; 5.2×1012) and AAV2/8-EF1α-DIO-RVG (BrianVTA; 4.2×1012) were injected into the PnO, and RV-EvnA-DsRed (BrianVTA; 2.1×108) was injected into the PnO. For optogenetic activation of LHA-PnO-spinal cord pathway in Fig. 3, AAV2/1-hSyn-FLP (BrianVTA; 1.5×1013) was injected into the LHA, and AAV2/2Retro-EF1α-ConFon-ChR2-mCherry (BrianCase; 4.0×1012) was injected into the spinal cord. For deletion of LHA-PnO or MSDB in Fig. 4 and Supplementary Fig. 4, AAV2/2Retro-EF1α-Cre (BrianVTA; 5.4×1012) was injected into PnO or MSDB, AAV2/9-EF1α-DIO-DTR-mCherry (BrianVTA; 6.0×1012) was injected into the LHA.

Optogenetic manipulation
Each optic fiber was attached to a 473 nm laser device (Shanghai Laser & Optics Century Co., Ltd, ADR 700A), and the laser pulses were triggered and stopped by a custom-written MATLAB code. For general optogenetic activation, a blue light stimulation with 473 nm a frequency of 10 Hz, and pulse duration of 40 ms was used. Laser power settings were adjusted for each mouse and maintained throughout all experiments. The power of the laser was between 10 and 40 mW at the connector. In the runway test, we recorded locomotion 3 s before stimulation, during the 3-s light pulse, and 3 s post-stimulation. For open field, elevated plus maze test, and food seeking test, each trial consisted of a 10-s light stimulation followed by a 20-s no-light interval.

Single-neuron projectome: data acquisition and analysis
Sparse labeling strategy. To achieve sparse and cell type-specific neuronal labeling across distinct neuropeptidergic populations, we employed a combination of conditional viral strategies in Cre-driver mouse lines targeting Adcyap1, Nts, Tac1, Tac2, Penk, Pdyn, SST, Oxytocin, MCH, Trh, CRH, and Orexin. Sparse labeling was accomplished either by injecting low-titer Cre-dependent AAVs expressing GFP variants or co-injecting Cre-dependent FlpO-expressing AAVs with FlpO-dependent fluorescent reporter AAVs at specific ratios. Additionally, for direct Orexin neuron labeling, we delivered AAVs encoding mNeoGreen under the control of the Orexin promoter (PPORX) into wild-type males. Together, these methods allowed us to achieve sparse labeling of multiple neuropeptide-defined neuronal subtypes with high specificity40.

fMOST imaging and preprocessing. As outlined in previous descriptions, the fMOST imaging process was carried out. Initially, the brains that had been dissected were subjected to post-fixation in 4% paraformaldehyde (PFA) before being embedded in Lowicryl HM20 resin (Electron Microscopy Sciences, 14340). Subsequently, the brains were imaged in a propidium iodide (PI) filled water bath using an fMOST microscope, achieving a voxel resolution of 0.32×0.32×1 μm. The surface of the sample in a coronal plane was then imaged to trace the neurons (GFP) and register the brain (PI channels), after which it was sectioned in 1 μm increments using a fixed diamond knife. This procedure, involving cycles of sectioning and imaging, was repeated until comprehensive imaging of the entire brain sample was accomplished.

We used a proprietary software tool, Fast Neurite Trace (FNT), to specifically trace long-range axon projections in the datasets produced by light microscopy. Initially, the software’s “slice2cube” function divides the original image data into more manageable 3D data cubes. At any given moment, approximately eight adjacent cubes around a specified position are automatically loaded into the computer’s memory and rendered in 3D for tracing. The tracing process consists of identifying a potential path, scrutinizing it, and expanding the existing neurite tree. User confirmation is required at each step to ensure the accuracy of the tracing process. This method allows for the comprehensive tracing of an axon projection. To ensure quality control, different neurons within the same sample were traced by different team members concurrently, with results cross-validated upon completion. Moreover, each traced neuron underwent an independent review by a separate individual to confirm accuracy. Additionally, we conducted random post-tracing quality checks on each brain sample.

The entire brain, complete with the information of all reconstructed neurons, was registered into the standard Allen CCFv3 using a method previously described. To summarize, we segmented several brain regions as landmarks, using cytoarchitecture references. We then executed a diffeomorphic transformation and symmetric image normalization using Advanced Normalization Tools (ANTs). This process enabled us to acquire transformation parameters based on these landmarks. Subsequently, these transformation parameters were applied to all the traced neurons within the brain sample. This allowed us to acquire transformation parameters based on these landmarks. Subsequently, these transformation parameters were applied to all the traced neurons within the brain sample. This allowed us to remap the reconstructed neuron onto the Allen brain template. The neurons with caudal projections that extend beyond the standard brain are thought to project to the spinal cord cervical c1 segment.

Neuron exclusion. We carried out manual checks on all neurons to confirm the accuracy of the tracing. Neurons that been found to have incorrect tracing data and with soma locations outside the lateral hypothalamus were excluded from subsequent analysis, after which a total of 916 neurons located in the LHA were incorporated in our analysis. All the neurons were mirrored to the same hemisphere prior to analysis.

Parameter calculation and neuron visualization. Utilizing the standard Allen CCFv3 annotation file as a reference, we computed the soma location, axon length, and terminal number for each neuron in every brain region. This was achieved using a Python library we developed, which is available at https://pypi.org/project/pyswcloader/. All neuron visualizations were generated using a self-developed Python package, neuron-vis (https://gitee.com/bigduduwxf/neuron-vis).

Hierarchical clustering of LHA neurons based on axon morphology. The projections and morphological information of LHA reconstructed neurons can be downloaded from our publicly accessible website (https://mouse.digital-brain.cn/projectome/lha).

To obtain the similarity matrix of axon morphology, first, we extracted the coordinates of each point from the original morphological file of each neuron, which were referred to by neuron P and Q, respectively. For each point i in the neuron P, we calculated the shortest distance to neuron Q and denoted this value as d(Pi→Q). The same calculation was performed on each point j of neuron Q to obtain the shortest distance to neuron P, which is denoted by d(Qj→P). Then we calculated a parameter α, the proportion of data points smaller than the mean value in each set of shortest distance, to get the weighted average value according to the value in each set of shortest distance, to get the weighted average value according to the following formula:

d(P→Q)=α*mean(d(Pi→Q))+(1-α)*max(d(Pi→Q))
d(Q→P)=α*mean(d(Qj→P))+(1-α)*max(d(Qj→P))

The similarity score between neuron P and Q was calculated as the mean value of d(P→Q) and d(Q→P). Then, hierarchical clustering using Ward’s linkage was performed on the similarity score matrix to identify the subtypes. The neurons projecting to the cervical spinal cord were assigned a fixed value.

DTR-mediated cell ablation. For local inhibition of the LHA, we injected Cre-dependent AAVs carrying diphtheria toxin receptor (DTR) into LHA of the Vglut2-Cre mice. For intersectional inhibition of PnO or MSDB projecting LHA neurons, we first injected AAV2-retro-Cre into the PnO or MSDB and then injected AAV-DIO-DTR-mCherry into the LHA of wild type mice. Three weeks after DTR injection, diphtheria toxin (DT, 50 μg/kg, Sigma) was intraperitoneally injected for three consecutive days to deplete the neurons. All the behavioral tests were conducted at least seven days after the DT injection. To confirm the effects of neuronal lesions, mice were sacrificed and perfused.

Chemogenetic experiments. Following at least 2 weeks for the expression of hM4Di, 2 mg/kg Clozapine N-oxide (CNO, BrainVTA) was injected intraperitoneally 1 h before behavioral tests. Prior to experiments, all animals received daily handling for 2 weeks to reduce stress-induced confounding factors associated with manual manipulation and injection procedures.

Optical-fiber-based Ca2+ recording in freely behaving mice. After 3 weeks of surgery, fiber photometry recording was carried out by using a commercial device (RWD Life Science, Shenzhen, China) as previously described. In brief, laser beams of 470 nm and 410 nm were initially launched into the fluorescence cube, then launched into the optical fibers. The 410 nm laser was used for motion control. The emission fluorescence from both the GCaMP and control was collected by a camera at the frequency of 20 Hz. The in vivo recordings were carried out within a custom-made open field box (40×40×40 cm), with food placed in a corner.

We calculated the value of the photometry signal, denoted as F, using the ratio F470/F410. We then computed ΔF/F as (F–F0)/F0, where F0 is the median of the photometry signal. Only behaviors with calcium signals exceeding 3 standard deviations (SD) were treated as events. The average of 10 peak ΔF/F and the number of events per minute for each mouse were analyzed.

Single unit recording and analysis. The 32-channel drivable electrodes were hand-assembled as previously described74. Custom tetrode arrays were manually fabricated, with 8-channel tetrodes mounted on a microdrive. Each tetrode was formed by twisting 13-μm polyimide-coated wires, and their tips were gold-plated to reduce impedance, resulting in a final impedance range of 500–800 kΩ (measured at 1 kHz). The tips of the tetrodes were implanted above the LHA and the electrodes were then fixed to the skull with dental cement. Recordings began five days after surgery, during which mice were allowed to move freely in a linear runway (1.5 m long × 15 cm wide × 20 cm high). After each recording, the electrodes were advanced by 100–200 μm. All signals were acquired using TDT system (Tucker-Davis Technologies, USA), digitalized and sampled at 24 kHz, then band pass filtered online (0.7–3 kHz). The stored data was converted and then processed using Offline Sorter for single-cell isolation (Plexon, Inc., TX, USA). The corresponding speed was obtained from synchronized videos recorded by the TDT system, which were analyzed using EthoVision XT (Noldus). The correlation between firing rate and speed was analyzed in a manner similar to previous work7.

EMG recording. For electromyography (EMG) recording, we performed implantation of customized bipolar electrodes into selected hindlimb muscles to record EMG activity. Electrodes (793200, A-M Systems) were guided by 30-gauge needles and inserted into the mid-belly of the medial gastrocnemius (GS) and tibialis anterior (TA) muscles of the hindlimb. A common ground wire was inserted subcutaneously in the neck-shoulder area. Wires were routed subcutaneously through the back to a small percutaneous connector, which was securely cemented to the skull of the mouse. EMG signals were acquired using a TDT system (RA32 pre-amplifier+RZ2 base processor) controlled by Synapse software (TDT) with a filtration range of 10–1000 Hz. The acquired signals were analyzed using custom-written MATLAB scripts.

Behavioral experiments and kinematic analysis. Motor functions were assessed weekly with a locomotor open field rating scale, the Basso Mouse Scale (BMS)75. All BMS behavioral tests were conducted by investigators who were blinded to the treatment groups.

Behavioral tracking of mouse movements was performed offline utilizing DeepLabCut (https://github.com/DeepLabCut/). Behavioral tests included overground locomotion of mice either prior to or throughout the recovery period following SCI on an elevated runway. More than 15% of the frames were selected from the behavioral videos of mice in different states to serve as the initial training datasets. To quantify the gait characteristics of mice before and after SCI, frames in the training datasets were manually labeled for the iliac crest, hip, knee, ankle, and toe. A ResNet-50 network was trained for 1×105 iterations on the initial training datasets, and the network with the lowest loss value was selected for subsequent analysis through evaluation. Videos representative of different mice were analyzed using the trained ResNet-50 network to obtain the labeled videos and the coordinates of the labeled points.

Kinematic variables were computed using custom scripts in MATLAB (v.R2018a, MathWorks), based on the x, y coordinates generated by DeepLabCut. The whole limb oscillation was evaluated by calculating the angle of the virtual limb linking hip to toe. The speed of toe was calculated by taking one frame from every six frames to determine whether the mouse was in the stance or swing phase, with the threshold being adjusted for each individual mouse. To visualize the movement of mice, we extracted a 3-s segment from the videos of different mice and displayed the labeled points and skeleton lines of their lower limbs.

Open-field (OF) test food seeking test and elevated plus maze (EPM). The test apparatus consisted of a rectangular box (40×40×30 cm). A dim light was applied above the field. Before the experiment, the mouse was put into the test room for a 30-min acclimation period. Subsequently, the mouse was gently placed into the center of the rectangular box for free exploration. The time spent in the center zone (20×20 cm), as well as the total distance and center time traveled in the whole open field arena, were measured over 20 min. For assessing the effect of BCI on reducing anxiety-like behavior in animals, animals in the BCI group triggered LHA-DBS using M1 signals. The control group was tested by providing stimulus pulses equivalent to the median number of pulses that trigger stimulation in the BCI group, with these stimulus pulses being applied either at regular intervals or randomly.

Specifically, for the group assigned to the task of food seeking, mice underwent a 24-h food restriction period prior to the test to enhance their motivation to food seeking during the assessment. During the fasting and testing period, they were maintained at more than 90% of their free-feeding body weight. Food pellets were placed in the center area and could be obtained by a single mouse in the open field for 20 min.

The EPM (Elevated Plus-Maze) apparatus used in our study consisted of a central region (5×5 cm), two open arms (30×5 cm), and two enclosed arms (30×5×15 cm). The apparatus, arranged in a “+” configuration, was situated 50 cm above the floor. At the beginning of the EPM test, the mice were placed in the central area, oriented towards an enclosed arm, and allowed to explore for a duration of 10 min.

In addition, we utilized EthoVision XT (Noldus) and custom-written MATLAB scripts to extract the total time that the animals spent in each zone of the open field or each arm of the EPM. Example trajectories were generated using EthoVision XT (Noldus). For both the open field and EPM test, we calculated the velocity by dividing the total distance by the moving time, data for which was also extracted using EthoVision XT.

SCI surgical procedures. The procedure for the T7 and T10 double lateral hemisection and T10 lateral hemisection was performed by a surgical technician. For the T7 and T10 double lateral hemisection, a T7-T10 laminectomy was made over the thoracic vertebrae. We utilized a scalpel and micro-scissors to disrupt the bilateral dorsal columns to make the T7 right-side over-hemisection, to make sure no sparing of the ventral pathways on the contralateral side. We carefully sectioned only the left side of the spinal cord up to the midline at T10. Subsequently, the muscle layers were sutured, and the skin was secured using wound clips. For the T10 lateral hemisection, a midline incision was made over the thoracic vertebrae followed by a T10 laminectomy. The unilateral hemisection was then performed carefully using both scalpel and micro-scissors, avoiding, to the greatest extent, the damage of the spinal cord dura.

Post-surgical treatments and care of the animals. Following SCI in adult mice, we administered a subcutaneous injection of 1 mL saline, positioned food on the floor of their cages, and ensured that they were kept warm until they regained consciousness. We undertook manual bladder emptying procedures twice a day and monitored for signs of dehydration and any clinical indicators of pain or infection. In case of a urinary infection, we initiated a course of antibiotherapy (Baytril, 10 mg/kg, for 5 days). Any mice exhibiting a body weight reduction exceeding 15% were humanely euthanized.

Deep brain stimulation. The deep brain stimulation (DBS) apparatus is composed of bipolar parallel tungsten wires, each with a diameter of 0.1 mm and a length of 5.5 mm. The insulation was removed at a length of 200 μm from the tip. The two electrodes were spaced 2.2 mm apart to bilaterally target the LHA. The DBS electrodes were secured to the head with dental cement. The mice were allowed to recover in their home cages for at least 3 days before starting the behavioral tests.

In the electrical stimulation mapping experiments, a stimulating electrode was lowered into the LHA under ketamine anesthesia. The electric current of −75 μA, 0.5 μs was delivered at a frequency of 45 Hz for 5 s, with intervals of 30–60 s between trials. The peak-to-peak amplitude and latency of the evoked responses were analyzed from EMG recordings of the right tibialis anterior (TA) muscle. Evaluations were conducted both with and without stimulation. The selected stimulation sites were strategically positioned along the AP axis of LHA at the coordinates of −0.54, −1.04, −1.54, −2.04, and −2.54 mm.

Intention-gated DBS. Two electrodes for recording and LHA stimulation were implanted under aseptic conditions and general anesthesia. To record the activities of M1 neurons, a 32-channel microelectrode array (4×8 array of tungsten wires, spaced 100 μm apart) was inserted into layer Ⅴ of the hindlimb area of the primary motor cortex (M1). In addition, a deep brain stimulation (DBS) apparatus was inserted in the LHA. The ground and reference wires from the array were attached to screws fixed to the skull.

During online testing, neural signals from M1 were monitored in real-time. Whenever the value of y=wn exceeded a detection threshold within the range of 0.7–0.8, the real-time processor administered LHA-DBS. Conversely, the DBS was halted when it fell to the threshold within the range of 0.2–0.327.

Immunohistochemistry and Imaging. Mice were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brain and spinal cord were removed and postfixed in 4% PFA at 4 °C for 24 h. The tissue was dehydrated in 30% sucrose solution at 4 °C for 24 h and sectioned coronally into 40 μm slices with a Cryostat microtome (Thermo Scientific HM525 NX). The sections were washed with PBS (6*10 min) and then blocked with a blocking solution (PBS containing 10% donkey serum and 4% Triton X-100) at room temperature for 2 h. Then the sections were incubated with the primary antibodies at 4 °C for 24 h. Upon completion of the primary antibody incubation, the sections were washed with PBS (6*10 min) and then incubated with secondary antibodies at room temperature for 2 h. After the 2-h incubation, the sections were thoroughly washed with PBS (6*10 min) and mounted with anti-fade mounting medium with DAPI for imaging. Sections were photographed using the Olympus VS120 virtual slide system.

Quantification and statistical analysis. All analyses were conducted blindly with respect to experimental conditions, using GraphPad Prism 10.0 software or MATLAB (v2018a). A two-tailed Student’s t-test was employed for single comparisons between two groups. For all other data, one-way or two-way ANOVA was utilized based on the appropriate experimental design. Post hoc analyses were performed exclusively when a main effect reached statistical significance, with P values for multiple comparisons adjusted using Bonferroni’s correction. All data are presented as the mean ± SD unless otherwise stated. ns, not significant; p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mice were randomized by litter, body weight, and sex before assignment to treatment groups; no additional specific randomization was implemented for the animal studies.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the present manuscript the authors utilize a wide range of methods to structurally and functionally trace the downstream projections of lateral hypothalamus (LH) that are involved in differently motivated locomotion. They then apply the found circuitry to selectively stimulate the spinal cord projections after spinal cord injury (SCI) and show that it can restore muscle activity and aid in recovery from injury.\nThe work is a comprehensive, and mostly well controlled investigation. Novelty is in describing the downstream circuitry by which LH can cause locomotion and functionally segregating the two downstream locomotion causing areas – medial septum and diagonal band of Broca (MSDB) and pontine oral part (PnO) – showing that MSDB is more involved in escape locomotive behaviours and PnO more in the food-seeking-motivated locomotion. Finally, authors show that optogenetic stimulation, DBS, and brain-controlled DBS of the LH-PnO-Spinal cord circuit in mice after SCI can acutely aid in mediating consequences of said injury. Furthermore, brain-controlled DBS assisted in recovery even when it was not switched on, showing long term functional restoration without causing anxiety associated with LH stimulation. The study represents an impressive collection of work, but before publication the study needs to address a couple of issues regarding data in support of their conclusions outlined below.\nMajor comments:\nIn Fig 3. a-e:\nThe authors show Open field test and Elevated plus maze tests for experiments selectively stimulating the LH-Vglut projections to MSDB or PnO, showing anxiety related behaviours are caused only by the MSDB projection stimulations.\nSubsequently, after selectively ablating the said projections using DTR, the authors ran a Food seeking test that showed impairment in centre entries (defined as food-seeking) only by ablations of PnO ablations.\nIt is strange the Food seeking test is not shown for the two optogenetic stimulation groups where one would expect PnO stimulation to potentially increase centre entries and MSDB to decrease. Similarly, the Open field test and Elevated plus maze test were not shown for the two ablated groups where one would expect changes after MSDB ablation only.\nThe authors should clarify if they ran these tests, and if so, show the results for a more comprehensive support for their conclusions about the functional segregation of these two projections in different types of locomotion. Understandably there is a caveat that escape behaviour was only triggered by stimulation, but a looming stimulus or sudden noise stimulus would trigger escape behaviours that could then implicate the LH-MSDB circuit in a deletion experiment. Alternatively, if the authors didn’t run these tests, they should explain why this was the case, including potential caveats in doing them, as it is a gap in the well-thought-out logical progression in the way the experiments were generally ran in the study.\nAdditionally, reduction of total movement in the LH-PnO ablation in Food seeking test could by itself cause fewer entries into the food area. It is difficult from just this to conclude that this circuit is functionally inclined toward motivated food seeking. The authors would need to provide an argument about the appropriateness of the given test given this caveat. For example, a good direct comparison would be with the same experiment without the food (open field).\nFurthermore, there is a lack of methodological description of optogenetic stimulations in these experiments whatsoever. The authors need to provide exact parameters of stimulation in these experiments, including descriptions of frequency, duration of stim, experiment length before and after stim that was used for measurements, number of repetitions etc.\nThere is also no description of statistical tests and number of animals for the experiments in b,c. The authors need to make sure here, and in other places in the manuscript, that information about statistical tests is neatly presented in figure legends.\nMinor comments:\nFig 4: in 4j height of lilac crest it used as a measurement to assess restoration of locomotor function, but in 4n maximal crest height is used. Authors should clarify whether this is a terminological inconsistency or intentional (with justification). Similarly maximal crest height is used in 6j.\nFormatting and grammatical errors should be fixed upon succesfull revision of the manuscript.", "reviewer_2": "In this article by Ji et al., to understand the role of lateral hypothalamic area (LHA) in the control of locomotion and its recovery after the partial spinal cord injury, the authors analyzed the single-neuron projectome of a large number of LHA neurons and classified them into 4 main clusters and 7 projectome-defined subtypes. Then, they optogenetically activated individual subclasses of neurons and found that the glutamatergic PnO and MSDB projecting neurons are related to regulation of locomotion. And particularly, the PnO projecting neurons regulate the locomotion via the reticulospinal neurons in the PnO without anxiety. Furthermore, the demonstrated that the PnO projecting neurons, which are mainly located in the caudal part of LHA, are involved in the spontaneous recovery of locomotion after incomplete spinal cord injury (SCI).\nFurthermore, they showed the DBS of caudal part of LHA promotes recovery after SCI and furthermore, that BCI based intention-gated DBS enhances recovery without affecting emotion of the subjects. Toward this conclusion, they conducted huge number of experiments. I think the authors should be admired for this achievement, but because they included descriptions on various experimental results, some parts are sparsely described and lack clarity. To improve the readability of this article, I would like to suggest a number of issues to be clarified.\nFig. 1j; difference between the green and gray bars is difficult to see.\nAnd in h, **** indicates p<0.001 and in l, it indicates p<0.0001. I recommend the authors to be consistent through the same figure.\nExtended Figure 1; the panel b does not include LHA. Why? The legend of panel d; what’s speed? Maximum speed or averaged speed during stimulation? Better to clarify in the legend.\nLine 113-114 of the main text; what are the criteria of glutamatergic and GABAergic neurons in LHA? The reference articles (34, 35) are on other brain regions.\nLine 136-142, the authors seem to have injected AAV-DIO-DTR into LHA of Vglut2+ mouse. Then, the effect of lesion should depend on the extent of diffusion of the vector. The authors should show that the lesion was confined to the intended area.\nLine 152-153 of the main text and Extended Figure 2a and b; “916 sparsely labelled neurons expressing various neuropeptides”; no description in the Method section about how the experiments were conducted.\nLine 189 of the main text; I am afraid that lack of stimulation effect of the spinal cord-projecting neurons (compared to PnO or MSDB-projecting neurons) was due to poor expression of ChR2 by the retrograde vector because the distance from the axon terminal to the cell body was longer. The authors should show that the spinal cord-projecting neurons strongly expressed ChR2 but the optogenetic stimulation did not cause the effect.\nLine 235 of the main text; this is just a comment. How do the authors think of different peptidergic neurons? Do they comprise another level of subpopulations of LHA neurons classified by the target regions?\nLine 272 Here the authors injected AAV-DIO-ChR2 into the spinal cord. Was the vector retrogradely transported to the cell bodies?\nLine 293-294 of the main text; here the authors mention that they injected at 3 days and 8 weeks after the injury, but Fig. 4b shows 1 week and 8 weeks. How does this difference come about? How ling was the survival time?\nLine 312 of the main text; “conditional genetic ablation of LHA glutamatergic neurons”; please explain more in detail about how this was done.\nLine 318 of the main text; can we tell that motivation was decreased just by decreased total distance for food seeking? These can just be caused by motor impairment.\nExtended Figure 4d and e; what is the difference from Figure 4d,g and h,k? I know the former is 2 weeks after injury and the latter is 1 week after injury, but what else?\nExtended Figures 5 and 6; can the authors show more enlarged views of the photomicrographs to show individual cells? It’s hard to see how the staining was changed in the current view size.", "reviewer_3": "This paper by Ji et al. investigates on the role of a glutamatergic population located in the caudal part of the lateral hypothalamic area (LHA) in the control of locomotion. Using single-neuron projectome analysis, transsynaptic and anterograde tracing, electrophysiological and imaging recordings, and electrical and optogenetic manipulations, the authors demonstrate that: (1) LHA neurons project downstream to PnO reticulospinal neurons that, which in turn reach the lumbar spinal cord; (2) specific activation of LHA-PnO glutamatergic neurons or their axonal terminals in the PnO elicits locomotion associated with food-seeking behavior; and (3) the locomotor activity evoked by optogenetic or electrical stimulation of the LHA-PnO-spinal cord pathway promotes the recovery of motor function in a mouse model of lumbar spinal cord injury.\nThis work is substantial and incorporates an impressive array of techniques and experimental perturbations. It presents potentially interesting results for a broad audience focused on locomotor control under physiological conditions, as well as for researchers with a more translational focus or those studying motor disorders. Although the study is, overall, technically and experimentally sound, there are several aspects that I believe should be addressed and further discussed. Some of these points are outlined below.\n1. The “Results” section requires thorough revision to improve the clarity and comprehensibility to this substantial body of work (see details below).\n2. As far as I understand, the locomotion induced by LHA optogenetic stimulation was performed in an open field (Fig. 2l) or in a linear corridor (videos). How do the authors demonstrate that the optogenetically evoked locomotion via the LHA-PnO-spinal cord pathway is motivational rather than merely regular locomotion?\n3. Line 317 and onward: Based on the data presented in this work, there is no doubt for this reviewer that activation of LHA neurons facilitates motor function recovery after spinal cord injury. However, it is not convincingly proved that ablation of LHA->PnO glutamatergic neurons specifically impairs motivational locomotion, as stated in line 326. Since the locomotion along the arena periphery is also reduced after DT ablation (Fig. 4), the observed decreases in total distance and centre entries may not be necessarily reflect a selective impairment of motivational locomotion. Rather, they could result from a decrease in the overall locomotor function. • Please discuss and clarify this matter.\nMore specifically:\nAbstract\n1. Line 25: “seeking via pontine oral part (PnO) projection neurons.”\nPlease replace it with: pontine reticular nucleus, oral part\nIntroduction\n2. Line 64: “the advantage of locally activating specific brain nuclei and has been successfully used to treat Parkinson’s disease23.”\nIn this work, Masini et al. did not “treat Parkinson’s disease”, they manipulated glutamatergic neurons in the PPN of a Parkinsonian mouse model to restore motor function.\nResults\n3. The text in the Results section requires revision, particularly in the first three parts of the section (lines 83-278).\na. The descriptions of the individual figure panels should be improved for clarity and completeness.\nb. The authors should specify the animal strains used in the different experiments and provide more detailed information about the recording methods or analytical approaches applied in each case.\nSome examples include:\n- Line 107: “we performed single-unit recording at the caudal part of the LHA while mice were walking on a linear track”. • Which recording method? and which mouse strain was used?\n- Line 109: “Analysis revealed a correlation between the firing rate and the speed, using a speed filter previously described”\n• Which type of analysis?\nc. The figure descriptions should follow the order of the panels or the sequence of diagrams/graphs within each panel/figure. Jumping back and forth in the narrative makes it difficult to follow and understand the results.\nd. Numerical results and statistical data should be explicitly referenced in the text.\n4. Line 88: “automatic analysis and registration of volumetric data from cleared tissues at 5 days after viral injection”.\n• However, in the Methods section under Retrograde-transsynaptic PRV tracing (line 730) it is stated: “For histological analysis, mice were perfused at 5.5 days after the PRV injection.”\n• Were the animals sacrificed 5.0 or 5.5 days after the injection?\n• How many synapses do the authors estimate the PRV could have retrogradely crossed in that time frame?\n5. Line 200: Fig 2j: Based on the figure, the MSDB is shown to receive inputs from the LHA “rostral projecting” neurons (labelled in red), yet the bar representing “Total distance” or the “Maximum speed” after the stimulation of LHA terminals in the MSDB is colored orange. Conversely, the “caudal projecting” label is orange, while the bar for LHA-PnO terminal stimulation is red. This inconsistency is potentially misleading. • Please swap the colors to ensure consistency between labels and the corresponding bars.\n6. Line 204: Activation of LHA-PnO projection terminals elicited low speed locomotion in contrast to LHA-MSDB which evoked high-speed responses.\nThis suggests that the LHA-PnO locomotor effect could be mediated via the LHA-PPN projections, given that low speed locomotion is controlled by the caudal PPN (Caggiano et al., 2018; Carvalho et al., 2020; Masini & Kiehn, 2022). • Have the authors considered this possibility? (See also comment #8)\n7. Line 205: “To further validate the involvement of the LHA-PnO and LHA-MSDB projections in locomotion, we injected retrogradely transported AAVs carrying Cre into the PnO and MSDB, respectively, while expressing Cre-dependent ChR2 in the LHA of wild type mice”\n• The authors have previously shown (line 105) that the LHA-PnO projecting neurons are glutamatergic. Why, then, did they use this approach in WT mice instead of using a Vglut-cre line, injecting a cre-dependent retro-ChR2 virus in the PnO, and implanting the optical fiber in the LHA? This alternative strategy would have provided greater specificity.\n8. Line 243 (Extended figure 3d): “Labeling of PnO-projecting LHA neurons revealed collateral axonal processes in the VTA and PPN”\n• Considering these projections, it is possible that LHA-induced locomotion could be mediated via glutamatergic neurons in the PPN. To rule out this possibility and support the claim that LHA-PnO neurons are “crucial” for evoking locomotion (as stated in line 273 and elsewhere), the authors should inhibit or ablate glutamatergic neurons in the caudal PPN and demonstrate that LHA stimulation still induces locomotion under these conditions. (See also related comments about in the discussion section)\nActivation of the LHA-PnO-Spinal pathway initiates locomotion\n9. Line 268: “We then expressed anterograde trans-synaptic AAVs (AAV serotype 1) carrying Cre into the LHA40-42, alongside AAV encoding Cre-dependent ChR2 in the PnO to stimulate the LHA-recipient PnO neurons” • In which animal strain?\n• This experiment demonstrates that activation of PnO neurons receiving input from the LHA can evoke locomotion (Fig. 3l); however, it does not stablish that the manipulated PnO neurons project to the spinal cord. (See next comment)\n10. Line 273: “To investigate whether PnO-spinal pathway directly controls locomotion, we injected retro AAV-DIO-ChR2 into the spinal cord of Vglut2- Cre mice. … (Fig.3m).”\n• The glutamatergic neurons retrogradely traced from the spinal cord are not necessarily the same population that receives input from the LHA (manipulated in the previous experiment, Fig. 3l). Therefore, the combined results of these two experiments (Fig. 3l and 3m) do not convincingly demonstrate that the LHA-PnO-SC pathway is directly involved in locomotion generation. To unambiguously prove the existence of a disynaptic LHA-PnO-SC pathway, the authors should inject in the LHA an AAV1 carrying Cre and a retro-Cre-dependent AAV carrying ChR2 into the spinal cord.\nLHA-PnO circuit contributes to spontaneous locomotor recovery following incomplete SCI\n11. Line 292: “Three groups of mice received PRV-injection from the hindlimb (GS/TA) muscle and sacrificed 5 days after injection…” • Which mouse strain?\n• Given that PRV is a polysynaptic, transsynaptic tracer—and considering the 5-day post-injection survival time—it is highly likely that the virus could have retrogradely spread to fourth- or even fifth-order neurons. • The authors should clarify how they ensured that the neurons labelled in the LHA following hindlimb injections are specifically part of the LHA-PnO-SC pathway, rather than components of a longer multisynaptic circuit.\n12. Line 311: “We performed conditional genetic ablation of LHA glutamatergic neurons in hemisection injured mice…”\n• Please state the method used to ablate the glutamatergic neurons.\n• In the current approach, all Vglut+ neurons in the LHA were ablated, not only just those located in the caudal portion or specifically projecting to the PnO. Therefore, it remains unclear if the observed decline in motor recovery is attributable specifically to the loss of LHA->PnO glutamatergic neurons, or whether it results from the broader ablation of the entire LHA glutamatergic population. The authors should clarify this point and, if possible, provide additional data or discussion to distinguish these two possibilities.\nAcute LHA stimulation enables stepping in paralyzed mice after stagger SCI\n13. Line 357: “We employed a staggered SCI model with opposite lateral hemisection at T7 and T10”\n• Could the authors explain why they use a different lesion model for this experiment compared to the lateral hemisection used in the previous lesion experiment (line 284)?\n• How comparable were the motor impairment phenotypes between the two lesion models?\nDiscussion\n14. Line 432: “Further electrophysiological mapping showed that stimulation of LHA or its surrounding areas52, 53 within SLR could induce locomotion after ablation of MLR2, 54.”\n• The authors cite two older reviews (Shink & Orlovsky, 1976 and Mori 1987) to support the claim that the SLR can evoke locomotion independently to the MLR. However, more recent studies have stablished that the glutamatergic neurons in the cuneiform (CnF) and the pedunculopontine (PPN) nucleus are those that initiate locomotion and control the speed (Roseberry et al. 2016, Caggiano et al. 2018, Josset et al. 2018, Carvalho et al. 2020). Partial ablation of the MLR does not prevent locomotion unless the lesion is bilateral and specifically targets the glutamatergic neurons in both nuclei that encompass the MLR (Caggiano et al, 2018). Furthermore, the present work shows that LHA neurons project to the PPN and not significantly to the CnF.\n•• Therefore, to demonstrate that the locomotor effects of LHA activation are independent of the PPN, it would strengthen the study if the authors ablated glutamatergic neurons in the PPN and showed that LHA glutamatergic activation still elicits locomotion under those conditions.\n15. Line 479: “Our study demonstrated that LHA Vglut2+ neurons projecting to the PnO play a crucial role in facilitating motivated locomotion, particularly during goal-directed behaviors such as food-seeking.”\n• The authors base this statement on two arguments:\na. Activation of LHA glutamatergic neurons evokes locomotion.\nThese experiments were performed in a linear corridor or an open field, were the mice exhibited locomotor bouts that were completely unrelated to food-seeking behavior.\nb. A reduction in the number of entries to the centre in the open field test (interpreted as food-seeking motivated locomotion) following Vglut+ neuron ablation.\nHowever, locomotion in the periphery of the arena is also reduced in these experiments (Fig. 4), which naturally decreases the total distance travelled and likely contributes to the reduced number of centre entries.\n••• In this scenario, could the authors elaborate on the arguments supporting the idea that the facilitation of food-seeking “motivated locomotion” is mediated by glutamatergic LHA-PnO neurons?\nTherapeutic potential of DBS and its implications for clinical translation in SCI\n16. Some reflections on the potential therapeutic advantages of LHA DBS compared to CnF or PPN stimulation would be a valuable addition to the discussion.", "reviewer_author_response": "Response to referees\nWe sincerely thank all reviewers and the editor for your time and constructive feedback.\nFollowing the reviewers’ suggestions, in this revised manuscript, we have revised the text, added\nnew analyses, and conducted additional experiments. Major changes are highlighted in yellow for\nclarity. We believe we have strengthened both the methodology and the conclusions of our study\nand hope the reviewers and the editor will find our revised work exciting and suitable for\npublication.\nReviewer #1 (Remarks to the Author):\nIn the present manuscript the authors utilize a wide range of methods to structurally and\nfunctionally trace the downstream projections of lateral hypothalamus (LH) that are involved in\ndifferently motivated locomotion. They then apply the found circuitry to selectively stimulate the\nspinal cord projections after spinal cord injury (SCI) and show that it can restore muscle activity\nand aid in recovery from injury.\nThe work is a comprehensive, and mostly well controlled investigation. Novelty is in describing\nthe downstream circuitry by which LH can cause locomotion and functionally segregating the two\ndownstream locomotion causing areas - medial septum and diagonal band of Broca (MSDB) and\npontine oral part (PnO) - showing that MSDB is more involved in escape locomotive behaviours\nand PnO more in the food-seeking-motivated locomotion. Finally, authors show that optogenetic\nstimulation, DBS, and brain-controlled DBS of the LH-PnO-Spinal cord circuit in mice after SCI\ncan acutely aid in mediating consequences of said injury. Furthermore, brain-controlled DBS\nassisted in recovery even when it was not switched on, showing long term functional restoration\nwithout causing anxiety associated with LH stimulation. The study represents an impressive\ncollection of work, but before publication the study needs to address a couple of issues regarding\ndata in support of their conclusions outlined below.\nWe sincerely appreciate Reviewer #1 for your thorough summary of our study and recognition of\nits achievements. We also thank your constructive suggestions to strengthen our conclusions.\nBelow, we have tried to address your concerns points by points.\nMajor comments:\nIn Fig 3. a-e:\nThe authors show Open field test and Elevated plus maze tests for experiments selectively\nstimulating the LH-Vglut projections to MSDB or PnO, showing anxiety related behaviours are\ncaused only by the MSDB projection stimulations.\nSubsequently, after selectively ablating the said projections using DTR, the authors ran a Food\nseeking test that showed impairment in centre entries (defined as food-seeking) only by ablations\nof PnO ablations.\nIt is strange the Food seeking test is not shown for the two optogenetic stimulation groups where\none would expect PnO stimulation to potentially increase centre entries and MSDB to decrease.\nSimilarly, the Open field test and Elevated plus maze test were not shown for the two ablated\ngroups where one would expect changes after MSDB ablation only.\nWe sincerely appreciate the reviewer’s insightful comments. In response, we have conducted the\nfollowing new experiments to address the behavioral effects of optogenetic stimulation and\nablation of the LHA-PnO and LHA-MSDB projections.\nOptogenetic Stimulation in the Food-Seeking Test (Extended Data Fig. 3a, b):\nWe performed optogenetic stimulation of the LHA-PnO pathway and found that it increased center\nentries without affecting food consumption, whereas stimulation of LHA-MSDB terminals\nsignificantly reduced center zone entries. These new results, presented in Extended Data Fig.\n3a, b, strengthened our conclusion.\nAblation Effects on Open Field and Elevated Plus Maze Tests (Extended Data Fig. 3c-e):\nWe performed new experiments and found that neither ablation of MSDB- nor PnO-projecting\nLHA glutamatergic neurons significantly altered locomotor activity in the open field test or\nanxiety-like behavior in the elevated plus maze.\nThese data (now included in Extended Data Fig. 3c-e of the revised manuscript) support our\nmodel that PnO projections primarily drive motivational-related locomotion (e.g., food-seeking),\nwhile activation of MSDB-projecting LHA neurons promotes anxiety-related behavior. We\nappreciate the reviewer’s insightful suggestion, which has helped clarify and solidify our\nconclusions (Line 298; Line 318).\nThe authors should clarify if they ran these tests, and if so, show the results for a more\ncomprehensive support for their conclusions about the functional segregation of these two\nprojections in different types of locomotion. Understandably there is a caveat that escape\nbehaviour was only triggered by stimulation, but a looming stimulus or sudden noise stimulus\nwould trigger escape behaviours that could then implicate the LH-MSDB circuit in a deletion\nexperiment. Alternatively, if the authors didn’t run these tests, they should explain why this was\nthe case, including potential caveats in doing them, as it is a gap in the well-thought-out logical\nprogression in the way the experiments were generally ran in the study.\nWe appreciate the reviewer’s insightful suggestion regarding the role of the LHA-MSDB circuit in\nnatural escape behaviors (e.g., induced by looming or auditory stimuli). While this specific\ninvestigation was not included in our current study, we would speculate that LHA-MSDB\nsilencing may not markedly attenuate natural escape responses, as such defensive behaviors could\nbe typically controlled by functionally redundant neural pathways. Indeed, previous work has\nshown that LHA-PAG glutamatergic projections mediate evasion behaviors, LHA-VTA\nglutamatergic inputs are required for defensive responses, and LHA-PPN projections coordinate\nsafety-directed locomotion1-3. Although our results clearly show that optogenetic stimulation of\nthe LHA-MSDB pathway robustly triggers escape-like running, demonstrating its sufficiency in\ndriving flight behavior, its necessity in escape contexts remains uncertain due to potential\ncompensation by these alternative evasion circuits. Thus, our findings primarily demonstrate that\noptogenetic activation of LHA-MSDB pathway is sufficient to drive escape-like locomotion,\nrather than proving it is necessary for escape contexts.\nAdditionally, reduction of total movement in the LH-PnO ablation in Food seeking test could by\nitself cause fewer entries into the food area. It is difficult from just this to conclude that this circuit\nis functionally inclined toward motivated food seeking. The authors would need to provide an\nargument about the appropriateness of the given test given this caveat. For example, a good direct\ncomparison would be with the same experiment without the food (open field).\nThe clear dissociation between locomotor patterns in the open field and food-seeking paradigms\nstrongly indicates that the LHA-PnO circuit specifically modulates motivation-driven locomotion,\nrather than overall locomotor activity.\nWe sincerely appreciate the reviewer’s insightful comment. To address the concern that reduced\nlocomotion per se might confound the interpretation of motivated food-seeking behavior, we\nconducted new open-field tests without food after ablation of PnO-projecting LHA neurons (now\nincluded as Figure 3).LHA-PnO ablation caused no significant change in total distance traveled in\nthe open field without food and this confirms that the reduced movement observed in the food-\nseeking test is not due to a global motor deficit. In contrast, when food was present in the center\nzone (food-seeking test), LHA-PnO-ablated mice exhibited decreased entries into the food zone\nand total distance travelled. The dissociation between locomotor activity in open field and food-\nseeking contexts strongly indicates that the LHA-PnO circuit specifically modulates motivation-\ndriven locomotion, rather than overall locomotor activity. We agree that locomotion and\nmotivation are often intertwined. However, the context-dependent nature of the behavioral deficits\n(present with food) supports our conclusion that LHA-PnO circuit is functionally specialized for\nmotivation-driven motivation. We have added this to the Discussion section (Line 683) and\nrevised the manuscript to clarify these points.\nFurthermore, there is a lack of methodological description of optogenetic stimulations in these\nexperiments whatsoever. The authors need to provide exact parameters of stimulation in these\nexperiments, including descriptions of frequency, duration of stim, experiment length before and\nafter stim that was used for measurements, number of repetitions etc.\nWe appreciate the reviewer’s suggestion about methodological description of optogenetic\nstimulation. We have now included comprehensive optogenetic stimulation parameters in the\nMethods Section (Line 1015). Below are the detailed specifications for clarity:\nEach optic fiber was attached to a 473 nm laser device (Shanghai Laser & Optics Century\nCo.,Ltd., ADR 700A) and the laser pulses were triggered and stopped by a custom-written\nMATLAB code. For general optogenetic activation, a blue light stimulation with 473 nm a\nfrequency of 10 Hz and pulse duration of 40 ms was used. Laser power settings were adjusted for\neach mouse and maintained throughout all experiments. The power of the laser was between 10-40\nmW at the connector. In the runway test, we recorded locomotion 3 seconds before stimulation,\nduring the 3-second light pulse, and 3 seconds post-stimulation. For open field and elevated plus\nmaze test, each trial consisted of a 10-second light stimulation followed by a 20-second no-light\ninterval.\nThere is also no description of statistical tests and number of animals for the experiments in b,c.\nThe authors need to make sure here, and in other places in the manuscript, that information about\nstatistical tests is neatly presented in figure legends.\nWe apologize for the oversight in our manuscript regarding the statistical details and sample size\ninformation. As suggested, we have now revised the figure legends to explicitly clarify:\nQuantification of center time and total distance of LHA-MSDB (top) or LHA-PnO (bottom)\nterminal stimulation in the OFT and EPM (n=6). One-way ANOVA/Tukey’s multiple comparison\nwas applied. ns, not significant; *p< 0.05;**p < 0.01; ***p< 0.001; ****p < 0.0001. Error bars,\nSEM.\nMinor comments:\nFig 4: in 4j height of lilac crest it used as a measurement to assess restoration of locomotor\nfunction, but in 4n maximal crest height is used. Authors should clarify whether this is a\nterminological inconsistency or intentional (with justification). Similarly maximal crest height is\nused in 6j.\nWe appreciate the reviewer’s attention to this detail. The \"height of lilac crest\" (Figs. 1p and 4j)\nand \"maximal crest height\" (Figs. 4n and 6j) indeed refer to the same measurement. This was an\noversight in terminology consistency, and we have revised all instances to explicitly use \"maximal\ncrest height\" for clarity throughout the manuscript.\nFormatting and grammatical errors should be fixed upon successfull revision of the manuscript.\nFormatting and grammatical errors have been carefully corrected in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nIn this article by Ji et al., to understand the role of lateral hypothalamic area (LHA) in the control\nof locomotion and its recovery after the partial spinal cord injury, the authors analyzed the single-\nneuron projectome of a large number of LHA neurons and classified them into 4 main clusters and\n7 projectome-defined subtypes. Then, they optogenetically activated individual subclasses of\nneurons and found that the glutamatergic PnO and MSDB projecting neurons are related to\nregulation of locomotion. And particularly, the PnO projecting neurons regulate the locomotion\nvia the reticulospinal neurons in the PnO without anxiety. Furthermore, the demonstrated that the\nPnO projecting neurons, which are mainly located in the caudal part of LHA, are involved in the\nspontaneous recovery of locomotion after incomplete spinal cord injury (SCI). Furthermore, they\nshowed the DBS of caudal part of LHA promotes recovery after SCI and furthermore, that BCI\nbased intention-gated DBS enhances recovery without affecting emotion of the subjects. Toward\nthis conclusion, they conducted huge number of experiments. I think the authors should be\nadmired for this achievement, but because they included descriptions on various experimental\nresults, some parts are sparsely described and lack clarity. To improve the readability of this\narticle, I would like to suggest a number of issues to be clarified.\nWe thank Reviewer #2 for your time and thoughtful comments. We have carefully considered all\nsuggestions and have clarified the corresponding text sections and performed additional analyses\nto improve the manuscript.\nFig. 1j; difference between the green and gray bars is difficult to see.\nAnd in h, **** indicates p<0.001 and in l, it indicates p<0.0001. I recommend the authors to be\nconsistent through the same figure.\nFollowing the suggestion, we have enhanced the color contrast between bars in Fig. 1j and\nstandardized the p-value markers throughout Figure 1, adopting *** for p < 0.001 and **** for p\n< 0.0001. These modifications have been incorporated into the revised manuscript.\nExtended Figure 1; the panel b does not include LHA. Why? The legend of panel d; what’s speed?\nMaximum speed or averaged speed during stimulation? Better to clarify in the legend.\nThe LHA data were originally presented in Figure 1e for clarity of the main findings. However, in\nresponse to this comment, we have now also included LHA in Extended Figure 1 (panel b) to\nensure complete representation and improve readability. In Extended Figure 1d, \"speed\" refers to\nthe instantaneous speed (individual time-point measurements) during stimulation, rather than\naverage or maximum speed. We have revised the figure legend to clarify this point. The updated\nversion now explicitly states: \"Instantaneous speed at each time point.\"\nLine 113-114 of the main text; what are the criteria of glutamatergic and GABAergic neurons in\nLHA? The reference articles (34, 35) are on other brain regions.\nWe appreciate the reviewer's insightful question. In our study, we identified these neuronal\npopulations based on the following electrophysiological characteristics:\nGlutamatergic neurons were defined by:\nTrough-to-peak duration ≥0.42 ms and baseline firing rate <5 Hz\nGABAergic neurons were characterized by:\nTrough-to-peak duration <0.42 ms and baseline firing rate ≥5 Hz\nWhile our initial references (34, 35) did indeed come from other brain regions, we agree with the\nreviewer that region-specific validation is crucial. We have now incorporated supporting reference\nwhich specifically established these classification criteria in the LHA. This reference has been\nadded to our revised manuscript to strengthen the foundation of our methodology (Line 123). We\nthank the reviewer for raising this important point.\nLine 136-142, the authors seem to have injected AAV-DIO-DTR into LHA of Vglut2+ mouse.\nThen, the effect of lesion should depend on the extent of diffusion of the vector. The authors\nshould show that the lesion was confined to the intended area.\nWe agree with the reviewer that clarifying the AAV-DIO-DTR-mediated lesion in the LHA of\nVglut2+ mice. To address this issue, we have added new immunohistochemical results (Extended\nData Fig. 1j) showing that mCherry expression was confined to the LHA. These results confirmed\nthat the observed functional effects were specifically caused by the targeted ablation of LHA\nVglut2+ neurons.\nLine 152-153 of the main text and Extended Figure 2a and b; “916 sparsely labelled neurons\nexpressing various neuropeptides”; no description in the Method section about how the\nexperiments were conducted.\nWe have accordingly updated the Methods section as follows:To achieve sparse and cell type-\nspecific neuronal labeling across distinct neuropeptidergic populations, we employed a\ncombination of conditional viral strategies in Cre-driver mouse lines targeting Adcyap1, Nts, Tac1,\nTac2, Penk, Pdyn, SST, Oxytocin, MCH, Trh, CRH and Orexin. Sparse labeling was accomplished\neither by injecting low-titer Cre-dependent AAVs expressing GFP variants or co-injecting Cre-\ndependent FlpO-expressing AAVs with FlpO-dependent fluorescent reporter AAVs at specific\nratios. Additionally, for direct Orexin neuron labeling, we delivered AAVs encoding mNeoGreen\nunder the control of the Orexin promoter (PPORX) into wild-type males. Together, these methods\nallowed us to achieve sparse labeling of multiple neuropeptide-defined neuronal subtypes with\nhigh specificity4 (Line 1027).\nLine 189 of the main text; I am afraid that lack of stimulation effect of the spinal cord-projecting\nneurons (compared to PnO or MSDB-projecting neurons) was due to poor expression of ChR2 by\nthe retrograde vector because the distance from the axon terminal to the cell body was longer. The\nauthors should show that the spinal cord-projecting neurons strongly expressed ChR2 but the\noptogenetic stimulation did not cause the effect.\nWe appreciate the reviewer's insightful comment. To address this concern, we performed\nsimultaneous retrograde co-labeling in the spinal cord using retroAAV-DIO-mCherry and\nretroAAV-DIO-EGFP revealed a high colocalization rate in LHA neurons, confirming efficient\nretrograde transduction. Our data demonstrated robust retrograde labeling and functional opsin\nexpression in spinal cord-projecting LHA neurons. The absence of behavioral effects appears to\nreflect the anatomical specificity of these connections, as LHA axons predominantly target dorsal\nspinal cord involved in sensory processing rather than ventral motor regions, consistent with their\nphysiological role in sensory modulation rather than motor control.These new results were\nsummarized as a figure below.\nLine 235 of the main text; this is just a comment. How do the authors think of different peptidergic\nneurons? Do they comprise another level of subpopulations of LHA neurons classified by the\ntarget regions?\nUsing Cre-driver lines targeting Adcyap1, Nts, Tac1, Tac2, Penk, Pdyn, SST, Oxytocin, MCH,\nTrh, CRH, and Orexin, we achieved sparse labeling of distinct neuropeptidergic populations (as\ndetailed in Methods Line 1027).\nLine 272 Here the authors injected AAV-DIO-ChR2 into the spinal cord. Was the vector\nretrogradely transported to the cell bodies?\nThank you for raising this technical issue. The viral vector used for optogenetic manipulation of\nspinal cord-projecting LHA neurons was retrograde AAV (retroAAV-CAG-DIO-ChR2-mCherry),\nwhich was injected into the spinal cord. The retroAAV is taken up by axon terminals and\nretrogradely transported to neuronal cell bodies. This adjustment has been made in the revised\nmanuscript (Line 379).\nLine 293-294 of the main text; here the authors mention that they injected at 3 days and 8 weeks\nafter the injury, but Fig. 4b shows 1 week and 8 weeks. How does this difference come about?\nHow ling was the survival time?\nWe appreciate the reviewer's careful observation. To clarify, while the first intervention was\nperformed 3 days post-injury, tissue collection occurred 5.5 days later. The text mentions\ninjections at 3 days post-injury and we elected to present this time point as \"1 week\" in Figure 4b\nas the standard convention for reporting experimental timelines.\nLine 312 of the main text; “conditional genetic ablation of LHA glutamatergic neurons”; please\nexplain more in detail about how this was done.\nConditional ablation of LHA glutamatergic neurons was achieved by bilaterally injecting AAV-\nDIO-DTR into the LHA of Vglut2-Cre mice. After 3 weeks for transgene expression, diphtheria\ntoxin was administered to selectively ablate Vglut2⁺ neurons.We have revised the text accordingly\nas follows: To selectively ablate LHA glutamatergic neurons, we bilaterally injected AAV-DIO-\nDTR into the LHA of Vglut2-Cre mice, followed by DT administration to induce targeted neuronal\nablation. After conditional genetic ablation of LHA glutamatergic neurons in hemisection injured\nmice, we measured basic over-ground locomotion and motivational vigor in fasting-triggered\nfood-seeking behaviors. We appreciate the suggestion and have made this adjustment in the\nrevised manuscript (Line 430).\nLine 318 of the main text; can we tell that motivation was decreased just by decreased total\ndistance for food seeking? These can just be caused by motor impairment.\nTo address the reviewer’s concern, we conducted open field test in mice with LHA excitatory or\nPnO-projecting neurons deletion after hemisection SCI (Extended Data Fig. 4d, f). These results\ndemonstrated that while locomotor recovery was impaired, there was no significant difference in\ngeneral locomotor activity compared to controls. In the food-seeking test, the significant reduction\nin both travel distance and number of entries indicates impaired motivation-related locomotor\nbehavior. The corresponding text has been updated accordingly. We sincerely appreciate the\nreviewer’s insightful comment, which has significantly strengthened the conclusions of our study\nand enhanced the clarity of data interpretation (Line 443; Line 465).\nExtended Figure 4d and e; what is the difference from Figure 4d, g and h, k? I know the former is\n2 weeks after injury and the latter is 1 week after injury, but what else?\nDuring spinal cord hemisection recovery, the early phase is critical for functional restoration.\nWe examined motor recovery and motivational locomotion following LHA glutamatergic neuron\nablation and PnO-projecting LHA neuron ablation.\nLHA glutamatergic neuron ablation: General motor recovery: Figure 4d; motivational\nlocomotion: Figure 4g (2 weeks after injury) and Extended Data Figure 4d (1 weeks after injury).\nPnO-projecting LHA neuron ablation: General motor recovery: Figure 4h; motivational\nlocomotion: Figure 4k (2 weeks after injury) and Extended Data Figure 4e (1 weeks after injury).\nExtended Figures 5 and 6; can the authors show more enlarged views of the photomicrographs to\nshow individual cells? It’s hard to see how the staining was changed in the current view size.\nWe sincerely appreciate the reviewer's suggestion regarding image clarity in Extended Figures 5\nand 6. In response, we have now included zoomed-in insets of representative regions from each\npanel to provide clearer visualization of individual cells at higher magnification.\nReviewer #3 (Remarks to the Author):\nThis paper by Ji et al. investigates on the role of a glutamatergic population located in the caudal\npart of the lateral hypothalamic area (LHA) in the control of locomotion. Using single-neuron\nprojectome analysis, transsynaptic and anterograde tracing, electrophysiological and imaging\nrecordings, and electrical and optogenetic manipulations, the authors demonstrate that: (1) LHA\nneurons project downstream to PnO reticulospinal neurons that, which in turn reach the lumbar\nspinal cord; (2) specific activation of LHA-PnO glutamatergic neurons or their axonal terminals in\nthe PnO elicits locomotion associated with food-seeking behavior; and (3) the locomotor activity\nevoked by optogenetic or electrical stimulation of the LHA-PnO-spinal cord pathway promotes\nthe recovery of motor function in a mouse model of lumbar spinal cord injury.\nThis work is substantial and incorporates an impressive array of techniques and experimental\nperturbations. It presents potentially interesting results for a broad audience focused on locomotor\ncontrol under physiological conditions, as well as for researchers with a more translational focus\nor those studying motor disorders. Although the study is, overall, technically and experimentally\nsound, there are several aspects that I believe should be addressed and further discussed. Some of\nthese points are outlined below.\nWe sincerely appreciate the reviewer’s constructive feedback, which have helped us improve the\nmanuscript. Below, we address each point in detail and have incorporated relevant modifications\nto clarify our findings.\n1. The “Results” section requires thorough revision to improve the clarity and comprehensibility\nto this substantial body of work (see details below).\nWe have carefully addressed these suggestions in the revised manuscript.\n2. As far as I understand, the locomotion induced by LHA optogenetic stimulation was performed\nin an open field (Fig. 2l) or in a linear corridor (videos). How do the authors demonstrate that the\noptogenetically evoked locomotion via the LHA-PnO-spinal cord pathway is motivational rather\nthan merely regular locomotion?\nWe sincerely appreciate the reviewer’s insightful comment. Our data mainly demonstrated that the\nLHA-PnO pathway promotes motivational locomotion, while PnO-spinal cord descending\npathway control basal locomotor function. We measured basic over-ground locomotion and\nmotivational vigor in fasting-triggered food-seeking behaviors, fasted mice exhibited increased\nentries and total distance indicating motivation-related locomotion (Fig. 1q, r).Ablation of LHA\nVglut2 or PnO-projecting LHA neurons selectively impaired food-seeking behavior (Fig. 1r and\nFig.3e, reduced center zone entries) while leaving basal locomotion in the open field test\ncompletely unaffected (Fig. 1q and Extended Data Fig.3 c-e). Additionally, we performed new\nexperiments of optical stimulation in food-seeking test and found that activation of LHA-PnO\npathway significantly increased center zone entries (40 trials in 20 min, each trial consisted of a\n10-second light stimulation followed by a 20-second no-light interval), indicating enhanced food-\nmotivated exploration (Extended Data Fig. 3a, b). These results support our conclusion that the\nLHA-PnO pathway functions as a neural substrate for translating internal drives (e.g., hunger,\nblood glucose) into motivation-directed locomotion. We have further clarified this important\ndistinction in the revised manuscript (Line 298; Line 318).\n3. Line 317 and onward: Based on the data presented in this work, there is no doubt for this\nreviewer that activation of LHA neurons facilitates motor function recovery after spinal cord\ninjury. However, it is not convincingly proved that ablation of LHA->PnO glutamatergic neurons\nspecifically impairs motivational locomotion, as stated in line 326. Since the locomotion along the\narena periphery is also reduced after DT ablation (Fig. 4), the observed decreases in total distance\nand centre entries may not be necessarily reflect a selective impairment of motivational\nlocomotion. Rather, they could result from a decrease in the overall locomotor function. • Please\ndiscuss and clarify this matter.\nTo address the reviewer’s concern, we conducted open field test after hemisection SCI.\nThe locomotor activity in the open field remained comparable among control, LHAVglut2-ablated,\nand PnO-projecting LHA neuron-depleted mice (Extended Data Fig. 4d, f) confirming that general\nlocomotion was equally impaired after SCI. However, fasted hemisection SCI control mice\nexhibited expected increase in food-seeking test (entries in food-accessible zones and total\ndistance), whereas ablation of either LHAVglut2 or PnO-projecting LHA neurons specifically\ndisrupted this motivational locomotion (Fig. 4g, k). Together, these results demonstrate that\nimpaired food-seeking reflects a deficit in goal-directed behavior—not general mobility—and this\nmotivational impairment correlates with delayed hindlimb recovery (Line 443; Line 465).\nMore specifically:\nAbstract\n1. Line 25: “seeking via pontine oral part (PnO) projection neurons.”\nPlease replace it with: pontine reticular nucleus, oral part\nIn response to the suggestion, we have modified the expression from \"pontine oral part\" to the\nmore accurate term \"pontine reticular nucleus, oral part\" (Line 25).\nIntroduction\n2. Line 64: “the advantage of locally activating specific brain nuclei and has been successfully\nused to treat Parkinson’s disease23.”\nIn this work, Masini et al. did not “treat Parkinson’s disease”, they manipulated glutamatergic\nneurons in the PPN of a Parkinsonian mouse model to restore motor function.\nWe sincerely appreciate your careful reading and professional critique. As you pointed out, Masini\net al.'s work focused on manipulating glutamatergic neurons in the PPN of a Parkinsonian mouse\nmodel to restore motor function, rather than directly \"treating Parkinson's disease\". We have\naccordingly revised the sentence to: “As a promising strategy for in vivo neuronal activation, deep\nbrain stimulation (DBS) offers the advantage of locally activating specific brain nuclei. Notably,\nMasini et al. successfully employed this approach to rescue motor deficits through targeted\nmanipulation of PPN glutamatergic neurons in a Parkinsonian mouse model.” (Line 64).\nResults\n3. The text in the Results section requires revision, particularly in the first three parts of the\nsection (lines 83-278).\na. The descriptions of the individual figure panels should be improved for clarity and\ncompleteness.\nb. The authors should specify the animal strains used in the different experiments and provide\nmore detailed information about the recording methods or analytical approaches applied in each\ncase.\nWe have revised the Results section to improve figure panel descriptions and clarify experimental\ndetails (animal strains, methods, and analysis). All changes are highlighted in the revised\nmanuscript. Specific modifications are detailed below.\nSome examples include:\n- Line 107: “we performed single-unit recording at the caudal part of the LHA while mice were\nwalking on a linear track”. • Which recording method? and which mouse strain was used?\nCustom tetrode arrays were manually fabricated, with 8-channel tetrodes mounted on a\nmicrodrive. Each tetrode was formed by twisting 13-µm polyimide-coated wires, and their tips\nwere gold-plated to reduce impedance, resulting in a final impedance range of 500–800 kΩ\n(measured at 1 kHz). Recordings were performed while C57BL/6 mice walked on a linear track\n(1.5 m long × 15 cm wide × 20 cm high). After each session, the tetrodes were advanced by ≥75\nµm to isolate new neuronal units in subsequent recordings. Following the final recording session,\nthe tetrodes were left in place and perfused for post hoc electrode position verification.This\nmethodology has now been explicitly detailed in the revised manuscript (Line 113; Line 1128).\n- Line 109: “Analysis revealed a correlation between the firing rate and the speed, using a speed\nfilter previously described” • Which type of analysis?\nThank you for pointing out the lack of clarity in our description. We analyzed changes in neuronal\nfiring rates in the caudal LHA in relation to speed using Spearman correlation analysis (to\nminimize resting-state contamination, we applied a speed filter excluding epochs below 5 cm/s).\nBased on your suggestions, we have revised the original text as follows (Line 117):\nTo further understand how the firing rate of LHA neurons relates to locomotion speed, we\nperformed single-unit recording at the caudal LHA of C57BL/6 mice walking on a linear track\n(1.5 m × 15 cm × 20 cm) using an 8-tetrode drivable array. A total of 156 neurons were recorded\nfrom 7 mice. Figure 1I shows an example neuron with firing rate positively correlated with speed.\nTo minimize resting-state contamination, we applied a speed filter (excluding epochs below 5\ncm/s) and found that 37 neurons (23.7% of recorded cells) exhibited firing rates significantly\nmodulated by locomotion speed (Spearman’s correlation, p< 0.01; median p = 0.42 (Fig. 1j).\nc. The figure descriptions should follow the order of the panels or the sequence of\ndiagrams/graphs within each panel/figure. Jumping back and forth in the narrative makes it\ndifficult to follow and understand the results.\nd. Numerical results and statistical data should be explicitly referenced in the text.\nWe thank the reviewer for this constructive suggestion. As requested, we have now explicitly\nintegrated key numerical results and statistical details into the main text.\n4. Line 88: “automatic analysis and registration of volumetric data from cleared tissues at 5 days\nafter viral injection”.\n• However, in the Methods section under Retrograde-transsynaptic PRV tracing (line 730) it is\nstated: “For histological analysis, mice were perfused at 5.5 days after the PRV injection.”\n• Were the animals sacrificed 5.0 or 5.5 days after the injection?\nThe correct time point for tissue collection was 5.5 days post-injection, and we have now\ncorrected it to ensure consistency (Line 91, Line 409).\n• How many synapses do the authors estimate the PRV could have retrogradely crossed in that\ntime frame?\nBased on established literature regarding the temporal progression of PRV-152 transsynaptic\ntracing5, 6, the estimated retrograde transsynaptic spread at 5.5 days post-injection typically covers\nfourth or even fifth synaptic steps including pyramidal system and extrapyramidal system in\nwhole brain. For the time course of PRV labeling, please also see in response 11.\n5. Line 200: Fig 2j: Based on the figure, the MSDB is shown to receive inputs from the LHA\n“rostral projecting” neurons (labelled in red), yet the bar representing “Total distance” or the\n“Maximum speed” after the stimulation of LHA terminals in the MSDB is colored orange.\nConversely, the “caudal projecting” label is orange, while the bar for LHA-PnO terminal\nstimulation is red. This inconsistency is potentially misleading. • Please swap the colors to\nensure consistency between labels and the corresponding bars.\nWe have standardized the color code for clarity: In the revised figure, we have standardized the\ncolor code for clarity: the LHA-MSDB (rostral-projecting) components are now uniformly\ndepicted in green, while the LHA-PnO (caudal-projecting) are consistently shown in red.\n6. Line 204: Activation of LHA-PnO projection terminals elicited low speed locomotion in\ncontrast to LHA-MSDB which evoked high-speed responses.\nThis suggests that the LHA-PnO locomotor effect could be mediated via the LHA-PPN\nprojections, given that low speed locomotion is controlled by the caudal PPN (Caggiano et al.,\n2018; Carvalho et al., 2020; Masini & Kiehn, 2022). • Have the authors considered this\npossibility? (See also comment #8)\nWe thank the reviewer for raising the important question regarding the potential involvement of\nPPN glutamatergic neurons in LHA-PnO pathway mediated locomotion3, 7-9. To directly test this\npossibility, we performed new experiments in which we silenced PPN glutamatergic neurons\n(using AAV-DIO-hM4Di in Vglut2-Cre mice with CNO administration) prior to optogenetic\nstimulation of the LHA-PnO pathway (Extended Data Fig. 2k-n). Importantly, we observed that\nlocomotion induction by LHA-PnO stimulation remained intact even during glutamatergic\nsilencing in PPN. These new findings provide evidence that the LHA-PnO pathway can initiate\nlocomotion independent of PPN glutamatergic activity. These modifications have been\nincorporated into the revised manuscript (Line 255).\n7. Line 205: “To further validate the involvement of the LHA-PnO and LHA-MSDB projections\nin locomotion, we injected retrogradely transported AAVs carrying Cre into the PnO and MSDB,\nrespectively, while expressing Cre-dependent ChR2 in the LHA of wild type mice”\n• The authors have previously shown (line 105) that the LHA-PnO projecting neurons are\nglutamatergic. Why, then, did they use this approach in WT mice instead of using a Vglut2-cre\nline, injecting a cre-dependent retro-ChR2 virus in the PnO, and implanting the optical fiber in the\nLHA? This alternative strategy would have provided greater specificity.\nIn response to this comment, we have now performed new intersectional experiments in Vglut2-\nCre mice by injecting retro-DIO-FLP into the PnO or MSDB and AAV-fDIO-ChR2 into the LHA,\nfollowed by optical stimulation in the LHA (Fig. 2 l-n and Extended Data Fig. 2i, j). Importantly,\nthis approach robustly induced locomotion, consistent with our original findings in WT mice.\nThese new data have been incorporated into the revised manuscript (Line 244) and further\nstrengthen the conclusions of our study. We thank the reviewer for this valuable suggestion, which\nhas significantly strengthened the precision of our experimental design.\n8. Line 243 (Extended figure 3d): “Labeling of PnO-projecting LHA neurons revealed collateral\naxonal processes in the VTA and PPN”\n• Considering these projections, it is possible that LHA-induced locomotion could be mediated\nvia glutamatergic neurons in the PPN. To rule out this possibility and support the claim that LHA-\nPnO neurons are “crucial” for evoking locomotion (as stated in line 273 and elsewhere),\nthe authors should inhibit or ablate glutamatergic neurons in the caudal PPN and demonstrate that\nLHA stimulation still induces locomotion under these conditions. (See also related comments\nabout in the discussion section)\nWe appreciate the reviewer’s insightful suggestion regarding the potential role of PPN\nglutamatergic neurons in mediating LHA-induced locomotion. To directly address this point, we\nconducted a new experiment to examine whether LHA-PnO pathway induced locomotion requires\nglutamatergic neurons in PPN. The PPN glutamatergic neurons were selectively silenced by\ninjecting AAV-DIO-hM4Di into the caudal PPN of Vglut2-Cre mice and administering CNO\nbefore locomotor testing. LHA-PnO optogenetic stimulation still evoked robust locomotion even\nwhen PPN glutamatergic activity was suppressed. These results demonstrate that LHA-PnO\nprojections can drive locomotion independently of PPN glutamatergic neurons, further supporting\nour conclusion that the LHA-PnO pathway is crucial for locomotion initiation. We have added\nthese data to the revised manuscript (Line 255).\nActivation of the LHA-PnO-Spinal pathway initiates locomotion\n9. Line 268: “We then expressed anterograde trans-synaptic AAVs (AAV serotype 1) carrying Cre\ninto the LHA40-42, alongside AAV encoding Cre-dependent ChR2 in the PnO to stimulate the\nLHA-recipient PnO neurons” • In which animal strain?\nThese experiments were conducted in wild-type (WT) mice (Line 373).\n• This experiment demonstrates that activation of PnO neurons receiving input from the LHA can\nevoke locomotion (Fig. 3l); however, it does not establish that the manipulated PnO neurons\nproject to the spinal cord. (See next comment)\nTo rigorously validate the involvement of LHA-PnOVglut2-Spinal cord in locomotion, we\nconducted complementary intersectional experiments in Vglut2-Cre mice (Fig. 3m). Specifically,\nwe injected AAV1-FLP into the LHA and Retro-ConFon-ChR2 into the spinal cord. (Also see next\nresponse)\n10. Line 273: “To investigate whether PnO-spinal pathway directly controls locomotion, we\ninjected retro AAV-DIO-ChR2 into the spinal cord of Vglut2- Cre mice. … (Fig.3m).”\n• The glutamatergic neurons retrogradely traced from the spinal cord are not necessarily the same\npopulation that receives input from the LHA (manipulated in the previous experiment, Fig. 3l).\nTherefore, the combined results of these two experiments (Fig. 3l and 3m) do not convincingly\ndemonstrate that the LHA-PnO-SC pathway is directly involved in locomotion generation. To\nunambiguously prove the existence of a disynaptic LHA-PnO-SC pathway, the authors should\ninject in the LHA an AAV1 carrying Cre and a retro-Cre-dependent AAV carrying ChR2 into the\nspinal cord.\nTo address this concern, we performed additional experiments in Vglut2-cre mice by injecting\nAAV1-FLP into the LHA and Retro-ConFon-ChR2into the spinal cord. Subsequent optogenetic\nstimulation in the PnO successfully evoked locomotion (new data in revised manuscript). This\napproach directly confirms that the LHA-PnOVglut2-SC pathway is involved in locomotion.We\nacknowledge this constructive suggestion and revised the text accordingly (Line 387).\nLHA-PnO circuit contributes to spontaneous locomotor recovery following incomplete SCI\n11. Line 292: “Three groups of mice received PRV-injection from the hindlimb (GS/TA) muscle\nand sacrificed 5 days after injection…” • Which mouse strain?\nThe mice used for PRV tracing were adult C57BL/6J wild-type (WT) mice. We have now added\nthe strain information to the Results section for clarity (Line 408).\n• Given that PRV is a polysynaptic, transsynaptic tracer—and considering the 5-day post-\ninjection survival time—it is highly likely that the virus could have retrogradely spread to fourth-\nor even fifth-order neurons. • The authors should clarify how they ensured that the neurons\nlabelled in the LHA following hindlimb injections are specifically part of the LHA-PnO-SC\npathway, rather than components of a longer multisynaptic circuit.\nTo ensure that the labeled neurons in the LHA are functionally relevant to the LHA-PnO-spinal\ncord pathway—rather than belonging to a longer, unrelated multisynaptic circuit, we performed\ntime-controlled viral tracing. Our results demonstrate a clear temporal progression of PRV\nlabeling:\n1.5 days post-injection: PRV-labeled neurons appear in spinal motor neurons (MNs) innervating\nthe hindlimb muscle, confirming initial viral uptake; 2.5 days post-injection: Labeling progresses\nto PnO neurons, consistent with their known monosynaptic input to spinal cord; 4 days post-\ninjection: PRV is detected in LHA neurons.\nFuture studies using monosynaptic retrograde tracers such as rabies virus could further refine the\nspecificity of this circuit. We are grateful for the reviewer's thoughtful suggestion.\n12. Line 311: “We performed conditional genetic ablation of LHA glutamatergic neurons in\nhemisection injured mice…”\n• Please state the method used to ablate the glutamatergic neurons.\nFor selective ablation of LHA glutamatergic neurons, Vglut2-Cre mice received bilateral LHA\ninjections of AAV-DIO-DTR, enabling Cre-dependent expression of the diphtheria toxin receptor\n(DTR). After 3 weeks for transgene expression, DT was delivered, resulting in the targeted\nablation of LHA Vglut2⁺ neurons. The relevant details have now been clarified in the revised\nmanuscript (Line 430).\n• In the current approach, all Vglut+ neurons in the LHA were ablated, not only just those located\nin the caudal portion or specifically projecting to the PnO. Therefore, it remains unclear if the\nobserved decline in motor recovery is attributable specifically to the loss of LHA->PnO\nglutamatergic neurons, or whether it results from the broader ablation of the entire LHA\nglutamatergic population. The authors should clarify this point and, if possible, provide additional\ndata or discussion to distinguish these two possibilities.\nWe sincerely appreciate the reviewer's insightful comment regarding the specificity of our LHA\nneuronal ablation approach. We performed specific ablation of LHA neurons projecting to the PnO\nand found that projection-specific ablation significantly impaired motor recovery after\nhemisection SCI, similar to the effects observed with Vglut2+ neuron ablation in the LHA (Fig.\n4h-j). These results strongly support the conclusion that the LHA-PnO pathway is critically\ninvolved in motor functional recovery.\nAcute LHA stimulation enables stepping in paralyzed mice after stagger SCI\n13. Line 357: “We employed a staggered SCI model with opposite lateral hemisection at T7 and\nT10”\n• Could the authors explain why they use a different lesion model for this experiment compared\nto the lateral hemisection used in the previous lesion experiment (line 284)?\nA staggered lesion was created at the T7 and T10 levels, comprising two lateral hemisections. The\nT7 lesion extended slightly beyond the midline, while the T10 lesion ended at the midline. This\ndouble hemisection paradigm deprived the lumbar spinal cord below the injury site of all direct\nbrain-derived innervation while leaving dormant circuits intact.The mice with this staggered\nlesion exhibited nearly complete and permanent hindlimb paralysis.The preserved intersegmental\nrelay circuits between the lesions make this model suitable for studying neuromodulation-based\nrecovery strategies. The manuscript has been revised accordingly (Line 520).\n• How comparable were the motor impairment phenotypes between the two lesion models?\nWe appreciate the reviewer’s question regarding the comparability of motor impairment\nphenotypes between spinal hemisection and staggered lesions.The hemisection model exhibited\nimmediate unilateral hindlimb paralysis on the injury side, with gradual recovery of locomotor\nfunction over time. In contrast, the staggered lesion animals demonstrated persistent, severe motor\ndeficits throughout the observation period, showing minimal recovery, suggesting that the spared\nrelay pathways between T7 and T10 remain dormant following injury10.\nDiscussion\n14. Line 432: “Further electrophysiological mapping showed that stimulation of LHA or its\nsurrounding areas52, 53 within SLR could induce locomotion after ablation of MLR2, 54.”\n• The authors cite two older reviews (Shink & Orlovsky, 1976 and Mori 1987) to support the\nclaim that the SLR can evoke locomotion independently to the MLR. However, more recent\nstudies have established that the glutamatergic neurons in the cuneiform (CnF) and the\npedunculopontine (PPN) nucleus are those that initiate locomotion and control the speed\n(Roseberry et al. 2016, Caggiano et al. 2018, Josset et al. 2018, Carvalho et al. 2020). Partial\nablation of the MLR does not prevent locomotion unless the lesion is bilateral and specifically\ntargets the glutamatergic neurons in both nuclei that encompass the MLR (Caggiano et al, 2018).\nFurthermore, the present work shows that LHA neurons project to the PPN and not significantly to\nthe CnF.\n•• Therefore, to demonstrate that the locomotor effects of LHA activation are independent of the\nPPN, it would strengthen the study if the authors ablated glutamatergic neurons in the PPN and\nshowed that LHA glutamatergic activation still elicits locomotion under those conditions.\nWe appreciate the reviewer’s insightful reference to the work by Roseberry et al. 2016, Caggiano\net al. 2018, Josset et al. 2018 and Carvalho et al. 2020, which highlights the caudal PPN’s role in\nlow-speed locomotion. In our revised manuscript, we have cited these works when discussing\npotential PPN-mediated mechanisms. While previous studies have highlighted the importance of\nthe PPN in locomotor control, our findings demonstrate that the LHA-PnO pathway can drive\nlocomotion independently of glutamatergic neurons in PPN. This conclusion is supported by our\nexperiments showing that optogenetic activation of LHA-PnO projections remains effective in\neliciting locomotion even when PPN glutamatergic neurons are silenced. Our work thus identifies\nthe LHA-PnO pathway as a distinct neural substrate for locomotor initiation. The corresponding\ncontent has been revised in the Discussion section (Line 663).\n15. Line 479: “Our study demonstrated that LHA Vglut2+ neurons projecting to the PnO play a\ncrucial role in facilitating motivated locomotion, particularly during goal-directed behaviors such\nas food-seeking.”\n• The authors base this statement on two arguments:\na. Activation of LHA glutamatergic neurons evokes locomotion.\nThese experiments were performed in a linear corridor or an open field, were the mice exhibited\nlocomotor bouts that were completely unrelated to food-seeking behavior.\nb. A reduction in the number of entries to the centre in the open field test (interpreted as food-\nseeking motivated locomotion) following Vglut+ neuron ablation.\nHowever, locomotion in the periphery of the arena is also reduced in these experiments (Fig. 4),\nwhich naturally decreases the total distance travelled and likely contributes to the reduced number\nof centre entries.\n••• In this scenario, could the authors elaborate on the arguments supporting the idea that the\nfacilitation of food-seeking “motivated locomotion” is mediated by glutamatergic LHA-PnO\nneurons?\nLHA-PnO ablation did not alter baseline locomotion (open field test), confirming that reduced\nmovement in food-seeking tests reflects motivation-specific impairment rather than general motor\ndysfunction. This context-selective deficit demonstrates the circuit’s specific role in translating\nmotivation into locomotor action. Conversely, stimulation of LHA-PnO enhanced motivation-\ndriven behavior, increasing food-approach entries.We thank the reviewer for raising this critical\npoint, and we have now incorporated further discussion of these context-dependent effects in the\nrevised manuscript (Line 683).\nTherapeutic potential of DBS and its implications for clinical translation in SCI\n16. Some reflections on the potential therapeutic advantages of LHA DBS compared to CnF or\nPPN stimulation would be a valuable addition to the discussion.\nWe sincerely appreciate the reviewer’s insightful suggestion regarding the potential therapeutic\nadvantages of LHA DBS compared to CnF or PPN stimulation. This is indeed an important\nconsideration, and we have expanded our Discussion to explicitly address this point (Line 745):\nOur findings raise important questions regarding the potential advantages of LHA-DBS compared\nto stimulation of other locomotor-related structures such as the MLR, which consists of the CnF\nand PPN. When compared to traditional DBS targets in the CnF and PPN, LHA stimulation\ndemonstrates several notable neurobiological and clinical advantages. Most significantly, LHA\ntargeting achieves robust motor improvements while substantially reducing the risk of stimulation-\ninduced adverse effects, particularly stress and anxiety. Furthermore, LHA stimulation appears to\nelicit more generalized motivational effects that synergistically enhance both volitional gait\ninitiation and adaptive locomotor control, whereas CnF/PPN stimulation tends to produce more\nstereotypic stepping patterns.\nReference\n1. Barbano, M.F., et al. VTA Glutamatergic Neurons Mediate Innate Defensive\nBehaviors. Neuron 107, 368-382 e368 (2020).\n2. Li, Y., et al. Hypothalamic Circuits for Predation and Evasion. Neuron 97, 911-924\ne915 (2018).\n3. Krauth, N., Sach, L.K., Sitzia, G., Clemmensen, C. & Kiehn, O. A hypothalamus-\nbrainstem circuit governs the prioritization of safety over essential needs. Nat Neurosci\n28, 1473-1485 (2025).\n4. Jiao, Z., et al. Projectome-based characterization of hypothalamic peptidergic\nneurons in male mice. Nat Neurosci 28, 1073-1088 (2025).\n5. Zhang, X., et al. Brain control of humoral immune responses amenable to\nbehavioural modulation. Nature 581, 204-208 (2020).\n6. Chan, K.L., Poller, W.C., Swirski, F.K. & Russo, S.J. Central regulation of stress-\nevoked peripheral immune responses. Nat Rev Neurosci 24, 591-604 (2023).\n7. Caggiano, V., et al. Midbrain circuits that set locomotor speed and gait selection.\nNature 553, 455-460 (2018).\n8. Masini, D. & Kiehn, O. Targeted activation of midbrain neurons restores locomotor\nfunction in mouse models of parkinsonism. Nat Commun 13, 504 (2022).\n9. Carvalho, M.M., et al. A Brainstem Locomotor Circuit Drives the Activity of Speed\nCells in the Medial Entorhinal Cortex. Cell Rep 32, 108123 (2020).\n10. Chen, B., et al. Reactivation of Dormant Relay Pathways in Injured Spinal Cord by\nKCC2 Manipulations. Cell 174, 521-535.e513 (2018)."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the raised concerns and discussed their implications in the revised manuscript. As such, the manuscript is recommended for publication.", "reviewer_2": "The authors carefully responded to my comments to the original version. I went through the revised manuscript and have no additional comments.", "reviewer_3": "The authors have made a substantial effort to respond to all my concerns and had conducted the suggested experiments to increase the specificity of some of the experimental manipulations. As a result, the pathway LHA-PnO-Spinal cord is now clearly defined after the intersectional experiments and shown to be independent of the caudal PPN glutamatergic neurons. Furthermore, the motivational nature of the locomotion triggered by LHA activation is more convincingly supported by the new ontogenetic activation experiment. These efforts have made the manuscript more robust and have strengthen the conclusions. I would like to congratulate the authors for their significant work.\nThe revised manuscript is now in much better form to be published in this journal, even though there are still some technical aspects that require clearer description, and I would like to discuss some details of the new experiments. Please see the points below.\n1. This study is a vast piece of work incorporating an impressive number of techniques, tests, and procedures. However, some of these tests or methodologies are either poorly described or entirely missing from the Methods section. Additional effort is required to improve these descriptions to allow reproducibility and to enhance clarity and understanding of the study.\n2. The new open field control experiment is a valuable addition, as it highlights the context dependent contribution of the LHA neurons to motivated locomotion (line 444 in the main text and Ext. Data Fig. 4d-g). However, I have some concerns about this experiment that I would greatly appreciate if the authors could clarify:\na. Were the animals of the open field experiment food-deprived, as they were in the food-seeking experiment (Ext Data 4)?\nCould the authors explain why the animals in control condition of the food-seeking experiment walk much more (~ 6000 cm; Ext Data 4e, g) than the controls in the open field test (~2500 cm; Ext Data Fig. 4d, f)?\nb. Why is locomotion in the center area of the arena quantified as “Center time %” in the open field test, but as “Center entries” in the food-seeking experiment? The same parameter (Center entries) should be used in both experiments to allow direct comparation.\nc. If the LHA>PnO neurons are described as “crucial”, so necessary and sufficient for evoking food-seeking motivated locomotion, their ablation should completely prevent such locomotion. However, the experiment shows only a decrease in food-seeking locomotion. Could this suggest that the ablation was incomplete?\nThis underscores the importance of validating the experimental approaches, in this case quantifying the number of ablated cells.\nMinor comments:\nLine 257: “The PPN glutamatergic neurons were selectively silenced by injecting…”\nPlease update to: “… silenced by bilaterally injecting…”\nLine 308: “retrogradely transported AAVs carrying Cre into the PnO while…”\nPlease update to: “… into the PPN or the MSDB, while…”\nLine 327: Please, remove the second “collectively”.\nLine 337: “. Interestingly, approximately 48.99% of neurons…”\nPlease update to: “approximately 48.99% of LHA neurons…”\nLine 423: “shown by electromyography recordings.”\nPlease clarify: Where are these recordings shown?\nLine 504: “these downstream brain regions during stimulation of…”\nPlease update to: “… during electrical stimulation of…”\nLine 667: Please update to: “… neurons are bilaterally suppressed.”\nLine 690-691: “motivation-driven motivation” Please, revise this phrase."}, "author_response": "Response to referees\nWe thank all reviewers and the editor for your time and valuable feedback, which has helped\nimprove our manuscript. All revisions are highlighted in yellow for clarity.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all the raised concerns and discussed their implications in the revised\nmanuscript. As such, the manuscript is recommended for publication.\nThank you for your positive evaluation and recommendation for publication. We are pleased that\nthe revisions addressed your concerns, and we appreciate your support of our work.\nReviewer #2 (Remarks to the Author):\nThe authors carefully responded to my comments to the original version. I went through the\nrevised manuscript and have no additional comments.\nWe sincerely appreciate your insightful review and confirmation that our responses addressed your\ncomments.\nReviewer #3 (Remarks to the Author):\nThe authors have made a substantial effort to respond to all my concerns and had conducted the\nsuggested experiments to increase the specificity of some of the experimental manipulations. As a\nresult, the pathway LHA-PnO-Spinal cord is now clearly defined after the intersectional\nexperiments and shown to be independent of the caudal PPN glutamatergic neurons. Furthermore,\nthe motivational nature of the locomotion triggered by LHA activation is more convincingly\nsupported by the new ontogenetic activation experiment. These efforts have made the manuscript\nmore robust and have strengthen the conclusions. I would like to congratulate the authors for their\nsignificant work.\nWe sincerely thank the reviewer for their positive feedback regarding our intersectional\nexperiments and characterization of the LHA-PnO-spinal pathway. We have now further discussed\nthese findings in the Discussion section (Line 683), further highlighting the pathway's specificity\nand its functional distinction from caudal PPN glutamatergic neurons. We appreciate the\nreviewer's valuable insights, which have helped strengthen our manuscript.\nThe revised manuscript is now in much better form to be published in this journal, even though\nthere are still some technical aspects that require clearer description, and I would like to discuss\nsome details of the new experiments. Please see the points below.\n1. This study is a vast piece of work incorporating an impressive number of techniques, tests, and\nprocedures. However, some of these tests or methodologies are either poorly described or entirely\nmissing from the Methods section. Additional effort is required to improve these descriptions to\nallow reproducibility and to enhance clarity and understanding of the study.\nWe sincerely appreciate your valuable suggestion regarding methodological clarity. As requested,\nwe have now thoroughly revised the Methods section to provide detailed descriptions of\ntechniques and procedures. These modifications are marked in the revised manuscript (Line 1032-\nLine 1068, Line 1167-Line 1172).\n2. The new open field control experiment is a valuable addition, as it highlights the context\ndependent contribution of the LHA neurons to motivated locomotion (line 444 in the main text\nand Ext. Data Fig. 4d-g). However, I have some concerns about this experiment that I would\ngreatly appreciate if the authors could clarify:\na. Were the animals of the open field experiment food-deprived, as they were in the food-seeking\nexperiment (Ext Data 4)?\nCould the authors explain why the animals in control condition of the food-seeking experiment\nwalk much more (~ 6000 cm; Ext Data 4e, g) than the controls in the open field test (~2500 cm;\nExt Data Fig. 4d, f)?\nWe sincerely appreciate the reviewer’s insightful question regarding the differences in locomotion\ndistance between the food-seeking and open field experiments. The animals in the food-seeking\nexperiment were food-deprived, which strongly motivated them to actively search for food,\nresulting in greater locomotion. In contrast, the open field test was conducted under normal\nfeeding conditions, leading to lower baseline locomotion in this less motivated state (Also see in\nFig. 1q and r).\nb. Why is locomotion in the center area of the arena quantified as “Center time %” in the open\nfield test, but as “Center entries” in the food-seeking experiment? The same parameter (Center\nentries) should be used in both experiments to allow direct comparation.\nWe sincerely appreciate the reviewer's constructive suggestion regarding the quantification of\nlocomotion parameters. Indeed, the open field test primarily assesses anxiety-like behavior, and\ncenter time % is widely accepted as a key metric reflecting anxiety levels in this paradigm (See\nreference 45. Zhang et al., Nat Neurosci. 2021). In contrast, the food-seeking experiment focuses\non motivation-driven locomotion and center entries serve as a more behaviorally relevant\nmeasure, directly reflecting locomotor activity associated with motivation-directed exploration\n(See reference 57. Li et al., Sci. Adv. 2021). Moreover, we fully agree with the reviewer that\ncross-experiment consistency strengthens data comparability. Thus, we have now incorporated\ncenter entries in the open field analysis (Fig. 6e, f and Extended Data Fig. 3a, e, Extended Data\nFig. 4d, f) (Line 286, Line 293, Line 328, Line 333, Line 455, Line 478, Line 581 and Line 589).\nc. If the LHA>PnO neurons are described as “crucial”, so necessary and sufficient for evoking\nfood-seeking motivated locomotion, their ablation should completely prevent such locomotion.\nHowever, the experiment shows only a decrease in food-seeking locomotion. Could this suggest\nthat the ablation was incomplete?\nThis underscores the importance of validating the experimental approaches, in this case\nquantifying the number of ablated cells.\nWe sincerely appreciate the reviewer’s insightful comment regarding the impact of LHA>PnO\nneuron ablation on food-seeking locomotion. While histological validation confirmed that ~80%\nof neurons were ablated (Extended Data Fig. 3g) (mCherry+ cells/section"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have addressed all my remaining concerns, and after a thorough revision process, I sincerely recommend the current version of the manuscript for publication.\nComment\nAs the authors clarified, the food-deprived animals have an additional drive (motivation) to move. Therefore, doesn’t this aspect make the locomotion performed under fasting conditions motivational per se, even in the absence of food? If that is the case, manipulation of LHA>PnO neurons should affect locomotion in this scenario, since the urge for food arises even though no food is present in the arena.\nAlthough this is a very interesting possibility to be explored, it does not warrant any further revision of the current work. However, the authors should consider addressing it in the discussion or in future studies.\nSuggestion\nExt. Data. Fig. 4\nSince the authors now show the number of “Entries to center area” for the two conditions — “Depletion of LHA glut neurons” (panels d, e) or “PnO-projecting LHA neurons” (f, g) — it would facilitate comparison if the graphs displaying the same data were aligned vertically, one above the other, as done for the “Total distance” graphs."}, "author_response": "Response to referees\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my remaining concerns, and after a thorough revision process, I\nsincerely recommend the current version of the manuscript for publication.\nComment\nAs the authors clarified, the food-deprived animals have an additional drive (motivation) to move.\nTherefore, doesn’t this aspect make the locomotion performed under fasting conditions\nmotivational per se, even in the absence of food? If that is the case, manipulation of LHA>PnO\nneurons should affect locomotion in this scenario, since the urge for food arises even though no\nfood is present in the arena.\nAlthough this is a very interesting possibility to be explored, it does not warrant any further\nrevision of the current work. However, the authors should consider addressing it in the discussion\nor in future studies.\nResponse: Thank you for raising this interesting point. Future studies will specifically test\nwhether fasting-driven locomotion, independent of food presence, engages LHA>PnO\nmotivational circuitry.\nSuggestion\nExt. Data. Fig. 4\nSince the authors now show the number of “Entries to center area” for the two conditions —\n“Depletion of LHA glut neurons” (panels d, e) or “PnO-projecting LHA neurons” (f, g) — it\nwould facilitate comparison if the graphs displaying the same data were aligned vertically, one\nabove the other, as done for the “Total distance” graphs.\nResponse: Both \"Entries to center area\" graphs are now vertically aligned for comparison."}]

Decision letter