Descending inhibitory rostral ventromedial medulla neurons cause widespread antinociception and contribute to the pain-inhibits-pain phenomenon

Domain: biology
Published: 2026-04-02
Editorial outcome: major_revision

Abstract
Acute painful stimuli applied to one body site reduce pain at other sites. The circuit basis of this “pain-inhibits-pain” phenomenon, also known as diffuse noxious inhibitory control (DNIC) in animals or conditioned pain modulation (CPM) in humans, is largely unknown. Using anatomical and optogenetic circuit tracing, we identified a population of descending inhibitory neurons of the rostral ventromedial medulla (RVM) that densely and bilaterally innervate the spinal cord along its rostrocaudal axis. Activating these neurons reduced heat and cold sensitivity widely in healthy mice and caused similarly wide-spread antihyperalgesia in chronic pain models, while their silencing evoked mechanical allodynia and spontaneous pain-like behaviors. Noxious stimuli activated subsets of these neurons in the lateral paragigantocellularis nucleus (LPGi), which inhibited nociception upon chemogenetic reactivation. Spinally projecting inhibitory RVM neurons are hence ideally positioned to function as circuit elements of DNIC and CPM, while their dysfunction may contribute to wide-spread chronic pain syndromes.

Introduction
The RVM, which includes the nucleus raphe magnus, the nucleus paragigantocellularis pars alpha (Pa) and the lateral paragigantocellularis nucleus (LPGi), serves as a critical hub for descending pain modulation systems 1, 2. Well-known examples of endogenous context dependent pain modulation include among others stress-induced analgesia and the pain-inhibits-pain phenomenon. Descending projections from the RVM are particularly important for context-dependent changes in conscious pain perception and can tune pain sensitivity in a bidirectional manner 3, 4. The RVM is the origin of several distinct descending fiber tracts that differ in their neurochemistry and anatomical organization. Previous work has identified neurotransmitters and neuromodulators released from descending RVM neurons, including glutamate, GABA and/or glycine, and serotonin, that are potentially involved in descending pain modulation. However, the precise anatomical organization of the underlying circuits and projections, and their contribution to specific forms of endogenous pain modulation are still incompletely understood 5-8 (for recent reviews see ref 9, 10).

It is well established that inhibition at the level of the spinal cord is mediated by the fast inhibitory neurotransmitters GABA and glycine, which tune nociceptive sensitivity to a physiologically meaningful level 11-13. In addition to local spinal interneurons, supraspinal projections (such as those from the RVM) provide a source of GABA and glycine to the spinal dorsal horn 3, 14. We and others have recently shown that the superficial layers of the dorsal horn, i.e. the sites where nociceptive afferent fibers terminate, are densely innervated by GABAergic and glycinergic axons originating from the RVM 3, 14, 15. It is hence conceivable that these projections contribute to context-dependent changes in pain sensitivity.

Here, we used intersectional approaches for high-precision anatomical and optogenetic circuit tracing to specifically investigate the spinal projections of inhibitory RVM neurons (vGAT RVMSC neurons). This approach was combined with chemogenetic and optogenetic activation and tetanus toxin mediated silencing to examine the biological functions of vGAT RVMSC neurons. We found that activating this circuit produced nearly body-wide, antinociception and antihyperalgesia via wide-ranging axons that project bilaterally throughout the entire length of the spinal cord. Experiments employing the Targeted Recombination in Active Populations (TRAP) technique demonstrated that a specific subset of these neurons, located mainly in the LPGi, were not only activated by noxious stimuli but also provided wide-spread analgesia upon chemogenetic reactivation. These projections therefore appear as critical elements of a circuit underlying the so-called “pain-inhibits-pain” phenomenon, also known as diffuse noxious inhibitory control (DNIC) in rodents and conditioned pain modulation (CPM) in humans. Accumulating evidence suggests that this control system is impaired in chronic pain conditions16, and interventions to restore it may have therapeutic potential. Accordingly, our silencing experiments indicate that compromised vGAT RVMSC neuron function causes pronounced mechanical sensitization to dynamic and punctate stimuli.

Results
Specific labeling of descending inhibitory RVM projections using AAV2retro vectors in vGATcre mice
To identify and manipulate neural pathways that descend from the RVM to the spinal cord, we used AAV2retro vectors to transduce RVM neurons via their axon terminals in the spinal dorsal horn 17. Since some projection neurons are resistant to retrograde transduction with these vectors 18-20, we first tested whether AAV2retro vectors would efficiently label the descending RVM neurons. We injected AAV2retro.eGFP into the left lumbar dorsal horn of 9 - 10-week-old mice and prepared tissue containing the RVM for multiplex fluorescent in situ hybridization (mFISH) (Fig. 1A). We found that many eGFP-labeled cells contained vGAT (19.4  4.1%) or vGluT2 (55.1  7.0%) mRNA, indicating that both excitatory and inhibitory neurons can be targeted with these vectors. Because the inhibitory neurons provide particularly dense innervation to the superficial dorsal horn3, 14, 15, we focused our analyses on this population. To specifically label these projections, we repeated the experiments in vGATcre mice and with a cre-dependent eGFP reporter construct. Most eGFP+ neurons in the RVM contained vGAT mRNA (83.6  9.3%) with only few containing detectable vGluT2 (2.0  2.0%), confirming eutrophic cre expression (Fig. 1B).

Since vGATcre is expressed not only in RVM projection neurons but also in interneurons of the RVM and spinal cord, we used an intersectional approach to specifically target those inhibitory RVM neurons that descend to the spinal cord 21, 22. To this end, we injected the lumbar spinal cords of vGATcre animals with AAV2retro vectors containing an optimized cre-dependent flippase (Flpo). One week later, the RVM was injected with a Flpo-dependent eGFP reporter construct (AAV9.dFRT.eGFPT) (Fig. 1C). To assess the accuracy and transduction efficiency of the RVM injections with AAV9.dFRT.eGFP, an mCherry sequence was included in the AAV2retro (AAV2retroA.flex.FLPo.mCherry) to directly label projections. We found that with single midline injections it was not possible to efficiently label vGAT RVMSC neurons located in the lateral and ventral RVM, such as the LPGi (Supplementary Fig. 1). We therefore switched to bilateral injections (-5.8, ± 0.5, 5.9 from Bregma), which reached more than 90% of the retrogradely labeled RVM projection neurons (Fig. 1C). This labelling strategy hence provided a reliable means of accessing most descending vGAT neurons across the RVM.

Global wide-ranging projections of vGAT RVMSC neurons
The spinal dorsal horn has a highly structured laminar organization, which is reflected by the segregated innervation of laminae by sensory fibers relaying different sensory modalities 23, 24. We therefore characterized the regional innervation pattern of the spinal cord by vGAT RVMSC neurons (Fig. 2A-C). We found that the axons of these neurons densely innervate the superficial laminae with only sparse innervation of the rest of the spinal cord (Fig. 2B). Strikingly, while labeling was performed from a single unilateral spinal cord injection site (left lumbar enlargement), labeled axon terminals were found bilaterally and throughout all spinal segments and including the spinal trigeminal nucleus of the brainstem (SPV) (Fig. 2B,C). Labeled axon terminals were also present in additional supraspinal sites (Fig. 2C), including the nucleus of the solitary tract (NTS), the lateral parabrachial area (LPb), and the periaqueductal grey matter (PAG). Notably, many of these areas are part of the ascending pain pathways 25, 26. To better visualize this projection pattern in its entirety, we used CLARITY-based tissue clearing and light sheet microscopy 27. These analyses revealed axons emanating from the RVM in both rostral and caudal directions and on both ipsilateral and contralateral sides (Supplementary video 1).

To quantify the extent to which vGAT RVMSC neurons projected to both sides of the spinal cord, we injected AAV2retro vectors containing cre-dependent mCherry into the left dorsal horn and cre-dependent eGFP into the right dorsal horn of the lumbar spinal cord (Fig. 2D). Although local spread of the AAVs was restricted to the injected side, many retrogradely labeled vGAT RVMsc neurons (31.0  1.5%) expressed both mCherry and eGFP, indicating that they projected bilaterally (Fig. 2D-F). Of those cell bodies that were labeled with only one fluorophore, most were found on the ipsilateral side (60.2  4.6%) (Fig. 2F). We also quantified the extent to which vGAT RVMSC project to different spinal cord segments. To this end, we retrogradely labeled vGATcre neurons from the cervical and lumbar spinal cords with different fluorophores (Fig. 2G). AAV2retro.flex.eGFP was injected into the lumbar cord and AAV2retro.flex.mCherry into the cervical cord. Many cells (39.7  2.2%) were labeled with both eGFP and mCherry, indicating that at least about 40% of the neurons provided bilateral inhibition over many spinal segments. Such wide-spread inhibition is further supported by the results of our optogenetic and electrophysiological experiments shown below. The remaining neurons were labeled with either eGFP (33.7  2.9%) or mCherry only (26.7  4.5%) (Fig. 2 H,I). An incomplete viral transfection efficacy may contribute to this finding. How ever, the behavioral data shown below that were obtained after tetanus toxin light chain-mediated silencing of vGAT RVMSC neurons support that the RVM also harbors descending vGAT neurons that provide more localized inhibition to the spinal dorsal horn.

Chemogenetic activation of descending inhibitory RVM projection neurons reduces nociceptive sensitivity
Our tracing experiments in vGATcre mice revealed dense innervation of the superficial dorsal horn, the first node in the ascending pain pathway. To assess the impact of these projections on nociception, we first performed chemogenetic activation experiments, introducing hM3Dq to the vGAT RVMSC neurons labeled from a unilateral site of the lumbar spinal cord using our intersectional strategy (Fig. 3A). The expression pattern of hM3Dq-mCherry in the spinal cord was bilateral (Fig. 3B) and virtually identical to that found in our previous labelling experiments. Upregulation of the activity-dependent marker c-Fos in retrogradely labeled RVM neurons verified the efficacy of the chemogenetic approach (Fig. 3C). Activation of these projections reduced the sensitivity of the ipsilateral hindpaw to heat and cold stimuli (Fig. 3D). In addition, activation reduced significantly the sensitivity to von Frey mechanical and pin-prick stimulation, indicating that they were capable of suppressing sensitivity to multiple sensory modalities. These changes in nociceptive sensitivity were not accompanied by detectable changes in responses to brush stimulation or in sensorimotor coordination assessed in the rotarod test. To validate that CNO-mediated effects depended on hM3Dq activation, we repeated the sensory assays in which mice had shown altered responses and compared the effects of CNO between animals expressing hM3Dq or, as a control, mCherry. We found significant increases in the withdrawal latencies and thresholds in the Hargreaves and cold plantar assays and electronic von Frey tests in hM3Dq-expressing animals relative to mCherry controls (Supplementary Fig. 2).

To verify that the antinociceptive effects described above depended on the activation of the descending spinal (rather than ascending brain) projections of vGAT RVMSC neurons, we followed two strategies. We first used local intrathecal injections of CNO (1 µg in 10 µl) or focal spinal optogenetic stimulation (0.2 ± 0.06 mW (irradiance = 1.72 mW / mm² applied at 20 Hz for 1s, every 10 s) of the lower lumbar spinal cord. Local CNO application increased withdrawal latencies from 9.55 ± 1.64 s to 19.11 ± 0.38 s (p = 0.002, paired t test, n = 5), and 2.12 ± 0.05 s to 7.95 ± 1.22 s (p = 0.008, paired t test, n = 5), for heat and cold, respectively (Supplementary Fig. 3 A – C). Focal optogenetic stimulation yielded similar results. Withdrawal latencies increased from 8.50 ± 0.92 s to 13.43 ± 1.11 s (p = 0.019, paired t test, n = 6), and 6.72 ± 0.40 s to 7.84 ± 0.30 s (p = 0.098, paired t test, n = 6), for heat and cold, respectively (Supplementary Fig. 3 D – E). Both experiments demonstrate that selective stimulation of the spinal terminals of descending vGAT RVM neurons alone is sufficient to induce analgesia. Secondly, we combined in vivo electrophysiological recordings of superficial dorsal horn with optogenetic stimulation of the spinal terminals of RVMSC neurons (Fig. 3 E,F). We expressed ChR2 from a viral transgene (AAV9.hEF1a.Dio.ChR2) in inhibitory (vGATcre) neurons of the RVM and performed extracellular recordings from wide dynamic range (WDR) neurons of the superficial lumbar dorsal horn. Action potential firing was reliably evoked through noxious or innocuous stimulation of the respective cutaneous receptive fields. Blue light stimulation of the lumbar dorsal spinal cord reduced action potential firing evoked by noxious stimulation by 79.3 ± 4.7% (n = 10, p = 0.0054, one-way repeated measures ANOVA). Action potential firing in response to innocuous stimulation was also reduced albeit to a smaller extent (45.3 ± 5.4%, n = 10, p = 0.043). In control experiments, when mCherry (rAAV9.EF1a.Dio.mcherry) was expressed instead of ChR2, blue light stimulation did not change stimulation-induced action potential firing (Supplementary Fig. 3 G,H). Taken together, these data indicate that activation of vGAT RVMSC neurons inhibits sensitivity to noxious thermal and mechanical stimuli through an action that involves descending inhibitory control of spinal nociceptive processing.

vGAT RVMSC projections form functional synapses throughout the spinal cord and can mediate widespread thermal antinociception
The presence of wide-ranging axon collaterals in multiple spinal segments raises the possibility that these neurons control nociception, not only bilaterally, but also along the rostrocaudal axis. To address this question, we again used an optogenetic approach and labeled descending vGAT RVMSC neurons with ChR2 from lumbar spinal cord injection sites and prepared slices from both the lumbar and cervical spinal cord of these mice. Subsequently, we tested whether light-evoked inhibitory postsynaptic currents (IPSCs) could be recorded both from lumbar and cervical superficial dorsal horn neurons (Fig. 4A). In agreement with the previous tracing experiments (Fig. 2), light-evoked IPSCs were present in both lumbar and cervical neurons. Light-evoked IPSCs from both locations were consistently blocked by co-application of bicuculline (20 µM) and strychnine (0.5 µM) (Fig. 4B,C). Separate application of either bicuculline or strychnine significantly reduced the amplitude of the light-evoked IPSCs (Supplementary Fig. 4), confirming the contribution of both transmitters.

We next asked whether this wide-ranging innervation would manifest in similarly wide-spread analgesia. We used the same expression strategy as above but introduced hM3Dq for chemogenetic activation (Fig. 4 D-G). First experiments addressing possible contralateral effects revealed significant changes in heat and cold sensitivity in the contralateral paw, but not in the von Frey filament assay in the contralateral paw (Supplementary Fig. 5). In subsequent experiments, we tested effects on heat and cold sensitivity in all four paws. In the Hargreaves test, we found significant CNO-induced latency changes for all four paws (Fig. 4E). In the cold plantar assay, heterotopic effects in the contralateral hindpaw and ipsilateral forepaw were significant but less pronounced (Fig. 4F). Finally, we tested whether the activation of the vGAT RVMsc neurons was also able to inhibit sensitivity of the facial areas to nociceptive stimuli. We applied droplets of the TRPV1 agonist capsaicin, which elicits burning pain sensations through activation of TRPV1 positive sensory neurons 28, to the eyes of mice. Nociceptive activation was quantified as the number of forepaw wipes directed towards the capsaicin exposed eye. Chemogenetic activation of descending vGAT RVMSC neuronsc traced from the lumbar spinal cord reduced the number of forepaw wipes when compared to vehicle-injected controls (Fig. 4G). Taken together, these data indicate that activation of descending vGAT RVMsc neurons inhibits nociceptive sensitivity in a near body-wide manner.

Silencing of vGAT RVMSC neurons evoked mechanical hypersensitivity, tactile allodynia, and spontaneous aversive behavior
Chemogenetic activation experiments demonstrated that excitation of descending vGAT RVMSC neurons reduces sensitivity to nociceptive stimuli, but whether these neurons also regulate baseline sensitivity remained to be explored. To this end, we performed loss-of-function experiments and used our intersectional approach to express tetanus toxin light chain (TetLC) in vGAT RVMSC neurons (Fig. 5A-C). Within seven days of virus injection, TetLC-expressing animals developed strong hypersensitivity to both punctate and dynamic mechanical stimulation (Fig. 5D). Strikingly, these animals exhibited spontaneous aversive behaviors, such as flinching of the ipsilateral hindpaw, and a reluctance to use this paw for locomotion (Supplementary video 2). Gross sensorimotor coordination assessed in the rotarod test remained unaltered. In contrast to the chemogenetic activation experiments, silencing the vGAT RVMSC neurons did not significantly alter heat or cold sensitivity, suggesting that these neurons are not involved in the control of heat and cold sensitivity in healthy mice. Although the observed changes largely affected the injected side, we also observed a slight increase in sensitivity to dynamic mechanical stimuli on the contralateral paw, consistent with our anatomical labeling experiment (compare Fig. 3).

These data suggest that vGAT RVMSC neurons include populations whose activation can inhibit nociceptive sensitivity, and other populations that are tonically active under resting conditions to control mechanical sensitivity. Accordingly, we hypothesized that there are neurons within the vGAT RVMSC population with lower basal activity that can be recruited to reduce nociceptive sensitivity, and others that have tonic activity and prevent mechanical hypersensitivity/allodynia. To address this, we performed targeted whole-cell recordings from retrogradely labeled vGAT RVMSC neurons (Fig. 5E-H). Recorded neurons could be broadly divided into three categories based on their spontaneous action potential firing patterns. These included spontaneously active neurons with regular or irregular firing, and inactive neurons (referred to as regular, irregular and silent respectively) (Fig. 5G), consistent with the findings of others29. Regular and irregular firing neurons could be distinguished based on the coefficient of variance of their interspike intervals (CV ISI) (Fig. 5H) 30. Regular firing neurons exhibited firing frequencies that varied between 3.2 and 14.7 Hz and were consistently higher than those of irregular firing neurons (0.13 - 0.77 Hz).

Recruitment of vGAT RVMSC neurons can reverse thermal and mechanical hypersensitivity
Silencing vGAT RVMSC neurons produced mechanical hypersensitivity that was reminiscent of that seen in pathological pain states. We therefore hypothesized that increased activation of these neurons could reverse hypersensitivity in inflammatory and neuropathic pain models. To test this possibility, we induced peripheral inflammation by injecting Complete Freund’s adjuvant (CFA) into the left hindpaw and repeated the chemogenetic activation experiments (Fig. 6A,B). Intraplantar injection of CFA increased mechanical and heat sensitivity, which was reversed by activation of vGAT RVMSC neurons (Fig. 6C). Strikingly, activation of vGAT RVMSC neurons was also sufficient to reverse CFA inflammation-induced hypersensitivity in the contralateral hindpaw (Supplementary Fig. 6). To determine whether a similar reversal of allodynia could also be achieved in pathological pain states, we performed chemogenetic activation experiments in mice with a chronic constriction injury (CCI) of the sciatic nerve 31. Activation of vGAT RVMSC neurons reversed mechanical allodynia measured on day 7 after nerve injury (Fig. 6D). Therefore, vGAT RVMSC neurons are not only required for normal mechanical sensitivity but are also able to reverse mechanical allodynia in hyperalgesic disease states.

vGAT RVMSC neurons are engaged during ongoing pain and provide feed-forward inhibition of nociception
Our data suggest that vGAT RVMSC neurons can broadly inhibit pain sensitivity throughout the body. We asked under what circumstances these neurons are engaged, and hypothesized that they might contribute to the so-called pain-inhibits-pain phenomenon. It is well-known that pain sensitivity can be reduced by other painful stimuli applied to a remote, e.g. contralateral site of the body. The involvement of descending inhibitory projections in this “pain-inhibits-pain” is well established 7, 32, 33. Neurons involved in this phenomenon are predicted to have at least three properties. They should be activated by painful stimuli, provide wide-spread analgesia, and be capable of reducing pain when active9. Since vGAT RVMsc neurons contain wide-ranging axonal projections and their activation reduces nociception, we next asked whether they are activated by pain. To address this, we labeled vGAT RVMSC neurons from the lumbar spinal cord and injected capsaicin into the right forepaw (20 µl capsaicin, 1 mg / ml) (Fig. 7A). We found that 17.8 ± 0.8% of labeled vGAT RVMSC neurons upregulated the activity-dependent marker c-Fos in response to capsaicin injection (Fig. 7B,C). Interestingly, these activated neurons were largely restricted to the LPGi (Fig. 7D).

To directly test if the same neurons activated by a painful stimulus provide wide-spread analgesia, we employed the Targeted Recombination in Active Populations (TRAP) technique and used TRAP2 (Fos2A-CreERT2) mice that express a tamoxifen dependent Cre recombinase under the transcriptional control of the activity-dependent marker gene c-Fos. We combined this approach with an intersectional viral strategy to restrict the expression of reporter or actuator transgenes to RVMSC neurons activated by noxious stimuli (Fig. 8A). We first characterized the neurochemistry of noxious stimulus activated RVMSC TRAP2 neurons (TRAP2caps RVMSC) using mFISH. To enable visual identification of TRAP2caps RVMSC neurons, we injected the lumbar spinal cord of TRAP2;Ai65 (cre and flp dependent tdTomato) double transgenic mice with AAV2retro.flex.FLPo. Two weeks later, capsaicin was injected into the right forepaw, followed by a 4-OHT injection one hour later. Another two weeks later, hindbrains from these animals were prepared for mFISH experiments (Fig. 8B). Most of the capsaicin-activated neurons contained vGAT (74.9%), a much higher proportion than non-specific labelling with AAV2retro.eGFP (Fig. 8C, see also Fig. 1A), indicating that most TRAP2caps RVMSC neurons were inhibitory. In agreement with the previous c-Fos labelling experiments (Fig. 7D), the majority of neurons were found in the LPGi (Fig. 8E). Also, in agreement with the previous labeling experiments, axon terminals of these neurons were most abundant in the superficial dorsal horn (Fig. 8F) and virtually all mCherry+ axonal boutons contained detectable levels of vGAT (Fig. 8G). When capsaicin was injected into the forepaw again, many (58.2 ± 2.5%) of the labeled neurons in the RVM upregulated c-Fos, indicating they were consistently activated by noxious stimuli (Fig. 8H,I). We then tested whether chemogenetic reactivation of the TRAP2caps RVMSC neurons would reduce nociception in the contralateral hindpaw, i.e., to a site distant from the initial stimulation (ipsilateral forepaw) (Fig. 8J). Sensitivity to noxious heat and cold stimuli was significantly reduced, while responses to mechanical stimuli were not altered. These findings demonstrate that vGAT RVMSC neurons in the LPGi are activated by noxious stimuli, and in turn reduce nociception in a wide-spread manner.

Methods
Animals
Mice aged between 6 - 12 weeks were used for behavioral and anatomical labelling experiments. For electrophysiology experiments, animals aged between 3 - 4 weeks were injected and were prepared for slice recordings >10 days later. Experiments were performed both in female and male mice. Different (opened and filled) symbols were used to identify data points obtained from female and male mice. Experiments were not powered to identify sex-specific differences. Additional experiments would be required to test whether such differences exist.

Permission to perform animal experiments was obtained either from the Veterinäramt des Kantons Zürich (154/2018 and 097/2021) or were approved by the National Institute of Dental and Craniofacial Research (NIDCR) ACUC and followed National Institutes of Health (NIH) guidelines. All transgenic mouse lines used in this study are listed in the Supplementary table 5. Animals were housed in a temperature- (21 - 24°C) and humidity- (40 – 60%) controlled facility on a 12h:12h inverted light cycle, with ad libitum access to food and water.

Experimental designs
Whenever possible, animals were randomly attributed to treatment group. When different genotypes were compared, we tested all mice in a certain litter with the correct genotype. In all behavioral experiments, the experimenter was blind either to the genotype of the animals or to their treatment.

Chemogenic experiments followed a cross-over design. Comparisons were made between the effects of CNO or vehicle treatment. In experiments involving tetanus toxin-mediated silencing of neurons, cre negative littermates were used as controls with the experimenter being blinded to the genotype. Comparisons were made before and 5 - 7 days after the RVM injection of AAVs containing the coding sequence for TetLc using a 2-Way ANOVA to test for a genotype * time interaction.

Virus injections
The dorsal horn of the lumbar spinal cord was injected as previously described 70, 71. Mice were anesthetized with 5% isoflurane which was maintained at 1 - 3% through a face mask during all surgeries. Analgesia (buprenorphine subcutaneously injected at 0.1 - 0.2 mg/kg) was provided before and after each surgery. During surgery, body temperature was maintained using a heated mat, and vitamin A cream was applied to protect the eyes. The skin above the injection site was shaved and disinfected with betadine. An incision was made to expose the T13 vertebra. The surrounding tissues were separated from the vertebral column and the T13 vertebra was clamped in position using spinal adaptors. A borehole was made in the middle of the T13 vertebra on one side and AAVs were injected into the spinal dorsal horn at a depth of 200 - 300 µm below the spinal surface approximately 400 µm lateral to the central artery. In some anatomical tracing experiments, both sides of the spinal cord were injected in a similar manner. For virus injections, 3 x 300 nl virus solution was injected along the rostrocaudal extent of the spinal cord at an infusion rate of 50 nl / min. The viral vectors used in this study are listed in the Supplementary table 3.

For injections into the RVM, the head was fixed in position using adjustable cheek bars. Injection coordinates were determined by the location of the retrogradely labeled RVM neurons that were identified in initial experiments, which relative to bregma were, and -5.8, ±0.5, 5.9 (rostrocaudal, mediolateral, dorsoventral respectively). Viruses were infused at 50 - 100 nl / min to a total volume of 1 µl for each injection. Injections were made using a motorized frame controlled by Neurostar Stereodrive software. The skull was installed in the frame to be as straight and as flat as possible (difference in z position between lambda and bregma < 100 µm). To compensate for variations in tilt and scaling, adjustments to the injection target were made in the software relative to four positions on the surface of the skull (Bregma, Lambda, 2 mm to the right of the midline, and 2 mm to the left of the midline).

Tissue preparation and immunohistochemistry
Mice were transcardially perfused with freshly prepared 4% paraformaldehyde (PFA) (room temperature, dissolved in 0.1 M phosphate buffer, adjusted to pH 7.4) following perfusion with 0.1 M phosphate buffer. Nervous tissues were quickly dissected and post-fixed in 4% PFA at 4C for two hrs. For CLARITY tissue clearing experiments, tissues were post-fixed for 24 hrs at 4°C after dissection (see below for more details). After post-fixation, brains or spinal cords were placed in 30% sucrose solution (dissolved in 0.1 M phosphate buffer) for 24 - 72 hrs for cryoprotection of the tissue. These were then rinsed with 0.1 M phosphate buffer before being embedded in NEG-50 mounting medium. Tissues were sliced at 60 µm on a sliding blade microtome (Hyrax KS 34, histocam AG) and stored as free-floating sections. For long-term storage, free-floating sections were stored in antifreeze medium (50 mM sodium phosphate buffer, 30% ethylene glycol, 15% glucose, and sodium azide (200 mg / l) at -20C until required.

For immunostaining, sections were first washed three times in 0.1M phosphate buffer and incubated in 50% EtOH for 30 minutes at room temperature, followed by three rinses in PBS with added salt (8 g NaCl per liter of PBS). Sections were incubated in a mixture of primary antibodies for two nights at 4C. The antibodies were reconstituted in a solution containing PBS with added salt, 0.03% Triton-X, and 10% normal donkey serum. All primary antibodies and dilutions used in this study are listed in the Supplementary table 1. Tissue sections were rinsed three times in PBS with added salt before being incubated in species specific secondary antibodies overnight at 4C (see Supplementary table 1). Finally, tissue sections were rinsed three times in PBS with added salt and mounted on microscope slides with Dako anti-fade medium.

Tissue clearing and light sheet microscopy
Following a 24-hour post-fixation in 4% PFA at 4C, tissues were placed in a pre-chilled hydrogel solution (containing 45 ml PBS, 5 ml 40% polyacrylamide, and 125 mg VA-044) sealed in an air tight container for > 2 days at 4C. Samples were baked at 37C for 3 hrs to allow polymerization of the hydrogel. Tissues were then transferred to an SDS-based clearing solution (containing 24.7g boric acid, 80g Sodium dodecyl sulfate in 2 l ddH O, adjusted to pH 8.5), and lipids were removed by active electrophoretic clearing for 3 - 5 hrs72. The clearing solution was circulated and chilled throughout the tissue clearing to avoid SDS warming and precipitation. Following active clearing, tissues were rinsed twice in PBS-T (containing 0.1% Triton-X) and then placed in histodenz solution overnight to allow for refractive index matching (refractive index adjusted to 1.4655 with PBS). Samples were glued to a platform and placed in an imaging cuvette filled with index-matched histodenz before being installed in a MesoSPIM light sheet microscope27. Images were acquired using MesoSPIM control and processed with ImageJ/FIJI.

Multiplex fluorescent in situ hybridization (mFISH)
For fluorescent multiplex in situ hybridization experiments, brains were quickly dissected following euthanasia (< 5 min). The hindbrains were isolated from the forebrain and cerebellum before being rapidly frozen in liquid nitrogen. Hindbrain tissues were embedded in Neg50 mounting media which was frozen for cryostat sectioning.

Tissue blocks were installed in a cryostat (Hyrax 60, histocam AG), and sections were taken at 20 µm thickness which were mounted directly onto superfrost microscope slices. Slides were stored at -80C before FISH experiments. All in situ hybridization experiments followed the guidelines according to the RNAscope Fluorescent Multiplex in situ hybridization v1 kit. Tissues were fixed for 15 min at 4C in freshly prepared 4% PFA dissolved in PBS. Following fixation, the slides were dehydrated through an alcohol series (50%, 70%, 100% and 100% again), for 5 min at each EtOH concentration. The tissue containing region of the slide was delineated with a heat-resistant fat pen and allowed to dry. Tissues were treated with a Protease solution (Protease IV) for 30 min at room temperature before hybridization. Protease was removed and slides were briefly rinsed twice in PBS, Z probes (ACD bio) were hybridized to the tissues for 2 hrs in a 40C purpose build oven. Probes were removed and slides were rinsed twice in wash buffer for 2 min each before and between all amplification stages. Amplification of the hybridized probes were achieved using the amplification reagents 1, 2, 3, and finally were either labeled with 4A, 4B, or 4C (for 30, 15, 30, and 15 minutes respectively) in a 40C oven. Slides were counterstained with DAPI for 10 minutes and mounted in DAKO antifade mounting medium. The probes used in this study are listed in the Supplementary table 2.

Cells were counted using a CellProfiler pipeline to identify nuclei with DAPI staining, and to assign fluorescent puncta from each detected mRNA species within a small area surrounding the nuclei. Cells were counted as positive if they contained above the 90th percentile of puncta/nuclei for each fluorophore, determined by running a 3-plex negative control on an adjacent slide in parallel with the same amplification reagents 73. Alternatively, experiments performed using the v2 version of the RNAscope Fluorescent Multiplex in situ hybridization kit, cells were counted manually using FIJI with the cell counter plugin.

Image acquisition and analysis
For an overview of injection sites and imaging entire brain sections, epifluorescence images were acquired using a Zeiss Axio Scan.Z1 slide scanner. For quantification of retrogradely labeled cells in the RVM, image stacks were acquired at 5 m z-spacing using a Zeiss LSM 800 confocal microscope. Confocal scans were made using 488, 561 and 640 nm lasers and the pinhole was set to 1 Airy Unit for reliable optical sectioning. Image stacks were acquired where immunoreactivity of all antigens was clearly visible. Acquisition settings were kept the same within each quantification experiment and acquired images were analyzed offline with FIJI using the cell counter plugin. Data were processed in Microsoft Excel and were presented and analyzed with GraphPad Prism 8.

TRAP2 labelling of activated neurons
To label projection neurons in the RVM that were activated by noxious stimuli, TRAP2 animals were used either in combination with an Ai65 reporter line, or by using an intersectional viral strategy. For the noxious stimulus, capsaicin (20 µl, 1 mg / ml) was injected into the plantar surface of the right forepaw 32. After one hour, 4 hydroxytamoxifen (4-OHT Sigma-Aldrich H6278) (1 mg / ml) was injected i.p. (10 mg / kg) for entry of the cre-ER into the nucleus to permit cre-dependent recombination. Stock solutions of 4-OHT were prepared at 40mg/ml in warm DMSO (50°C) and were kept in aliquots at -20°C. To dissolve 4-OHT in an aqueous solution, saline containing 2% Tween 80 and 2.5% DMSO, and was warmed to 50°C during preparation of the working solution 74. Experiments were performed at least ten days following the final stimulation paired with 4-OHT. One or two stimulation 4-OHT pairings were performed to efficiently label as many pain-activated neurons as possible.

Behavioral assays
For activation or silencing of descending RVM projections, we used an intersectional strategy in which an AAV2retro.flex.FLPo virus (also containing either a BFP or mCherry coding sequence) was used to transduce descending cre-expressing neurons. One week later, mice received RVM injections of AAVs containing a FLPo-dependent effector (either hM3Dq or TetLC). For DREADD experiments, 10 days incubation time was given to allow the expression of the receptor, whereas 5 - 7 days were given for TetLC expression before sensory and motor testing. Before experiments mice were acclimatized to the behavioral setup for at least one hour. For the Hargreaves, cold plantar, electronic von Frey, and Rotarod assays, an average from six measurements was recorded per time point. For brush and pin prick tests, an average from 10 response scores was recorded per time point. All measurements were taken from both hindlimbs of all animals, and in some experiments, the forepaws were also tested, using the same scoring criteria for testing hindpaws.

For behavioral testing of TRAP2 mice, three experimental groups were used. (i) pre-TRAP, TRAP2 animals that received injections depicted in Figure 8D, before the capsaicin forepaw injection and/or 4-OHT delivery. (ii) post-TRAP, animals that received the injections shown in Figure 8D at least 10 days after forepaw capsaicin injection and 4-OHT. (iii) control-TRAP, animals that received the injections in Figure 8D, 10 days after 4-OHT delivery but with no forepaw capsaicin injection.

Hargreaves test
To measure heat sensitivity, paws were stimulated with an infrared heat source (Hargreaves plantar assay IITC). Mice were placed on a transparent platform preheated to 30C, and withdrawal latencies were recorded in response to heating from an infrared heat source using an inbuilt timer. The stimulation intensity was set to 20% heater power for each test, and a cutoff time of 32 s was used to prevent potential tissue damage, with 3 - 5 min between stimulating the same paw to avoid tissue sensitization.

Cold plantar assay
Mice were placed on a 5 mm borosilicate glass platform and were stimulated from beneath with dry ice pellets. The time taken between touching the glass from below to the withdrawal of the paw was measured and 20 s was used as the cutoff time to avoid any tissue damage. The same paw was only tested again after a 3 - 5 min recovery time.

Electronic von Frey
Mechanical thresholds were determined using an electronic von Frey algesiometer (IITC). Animals were adapted on a mesh surface, and the plantar surface of each paw was stimulated with a bendable plastic filament attached to a pressure sensitive probe. Pressure was applied to the plantar surface gradually at a constant speed until the animal withdrew its paw. The maximum pressure that was present when the animal withdrew its paw was recorded.

Dynamic allodynia (brush) test
Sensitivity to dynamic mechanical stimuli was a measured by gently brushing the hindpaw plantar surface with a paintbrush at a constant speed from heel to toe. Response scores were given for each stimulation and were based on methods previously described, with some adaptations to the scoring 75, 76. In brief, animals that did not withdraw their paw were given a score of 0, and animals that quickly withdrew were given a score of 1. Animals that withdrew and kept the paw raised (extended lifting) for > 0.5 s were given a score of 2, and animals that flinched or licked the paw post-stimulation were scored as 3. Repeated flinching and licking of the stimulated paw were given a score of 4.

Manual von Frey test
Von Frey thresholds were determined using a set of calibrated von Frey filaments and a simplified version of the up-down method. In brief, the glabrous skin of the plantar surface of the paw was stimulated for 3 -5 s starting with the 0.4 g filament. Gentle pressure was applied until the filament started to bend, and each filament was used up to 5 times. A positive response was recorded when the mouse withdrew or flinched its paw, and a negative response was a lack of paw withdrawal. If three responses were positive the next lighter filament was chosen, and if there were three negative responses the next higher weight filament was used. This process was repeated five times, and the 50% withdrawal threshold was calculated for each paw.

Rotarod
Sensorimotor coordination was evaluated using an accelerating rotarod, and the time taken for animals to fall from the rotating barrel was recorded. The barrel rotated from 4 - 40 rpm accelerating at a constant rate over a period of 300 s. Values were not included if the animal jumped from the barrel in the direction of rotation, and if the animal jumped in > 50% of trials for a given time point these data were discarded.

CFA inflammation
To induce inflammatory hyperalgesia, complete Freund’s adjuvant (CFA) (Sigma Aldrich F5881) was injected into the plantar surface of the ipsilateral hindpaw. Under anesthesia (5% isoflurane for induction, 1 - 2% for maintenance) 20 µl of CFA was injected subcutaneously into the plantar surface of the paw. The needle was left in place for 5 - 10 s after injection to reduce backflow. Mice were tested for up to 2 days following the injection.

Chronic constriction injury model
Neuropathic pain was induced by applying a chronic constriction injury (CCI) to the left sciatic nerve proximal to its trifurcation. Three loose ligatures were put around the sciatic nerve, using 5-0 surgical silk, while mice were anesthetized with isoflurane 1 - 3%. The skin was closed with 2 - 3 sutures. Mice were tested on day 7 after CCI surgery.

Capsaicin eye assay
Mice were adapted to a cylindrical plexiglass chamber for 15 min. Animals were restrained and 10 µl capsaicin (0.1% in 5% Tween 5% ethanol PBS) or vehicle was dropped onto the eye. Mice were returned to the chamber and the number of forepaw wipes directed to the affected eye were counted within the following 3 min.

Chemogenetic experiments
Stock solutions of CNO were stored at a 500x concentration in DMSO at 100 mg / ml. Immediately prior to injection, CNO was diluted in sterile filtered saline. For chemgenetic experiments, CNO was injected either i.p. (2 mg / kg) or intrathecally (at 1 µg in 10 µl). In i.p. injection experiments, animals were tested directly before and 1 - 3 hrs after i.p. injections. For intrathecal injection, animals had the hair above the lumbar spinal segments shaved and removed with hair removal cream at least one day before behavioral experiments. Mice were briefly anesthetized with 2-3% isoflurane (< 2 min) and placed on a circular falcon tube to expose the intervertebral spaces. The T13 vertebra was identified, secured, and 10 µl CNO (1 µg in 10 µl) was injected intrathecally with a 29G insulin syringe. Injection sites were confirmed by the presence of a tail flick reflex during injection. Animals were returned to the testing arena, and sensory testing was performed at least 15 min after animals regained consciousness.

In vivo optogenetic stimulation for behavioral experiments
Eight to 10 week-old vGATcre mice were implanted with spinal cannulas for local light delivery as described previously 77. Implantations were made two weeks after brain injection of 1µl of AAV9.flex.hChR2_EYFP bilaterally in the LPGi. In brief, mice were anesthetized with 5% isoflurane and kept under anesthesia with 2-3% isoflurane on a stereotaxic frame throughout the duration of the surgery. After shaving the fur on the back of the mouse, a skin incision was made to expose the vertebral column and cuts were performed on the muscles located medially to the longitudinal tendons running along the vertebral bodies, to better expose the vertebrae. The vertebral column was subsequently clamped at the level of the thirteenth thoracic vertebra (T13). After carefully removing connective tissue and absorbing excessive bleeding with collagen strips (Lyostypt, B. Braun), a hole was drilled approximately 1 mm left of the spinous process of the T13 vertebra. After placing the cannulas (Doric lenses) with ferrules (⌀ 1.25 mm) on the drilled hole, two layers of 3M™ Scotchbond™ Universal Self Etch Adhesive were applied and cured with UV light for 10 s each to fix the cannula in place. To ensure a more stable implant, Tetric Evoflow® A1 (Ivoclar vivadent) was subsequently applied and also cured with UV light for 10 s. The muscles along the vertebral column were then sutured using absorbable sutures (Safil 5-0, B. Braun). The skin was sutured with non-absorbable threads (Dafilon 6-0, B. Braun). Mice were allowed to recover on a heat pad. Behavioral experiments were conducted 10 days after surgery.

To measure optogenetically-evoked behavior, mice were placed in plexiglass cylinders (10x10 cm wide, 25 cm high). Immediately before sensory testing, cannulas were connected through a mating sleeve to the optic fiber (0.39 NA multimode fiber-optic patch cable, Thorlabs, Inc) with a rotatory joint to allow free movement of the mouse. The fiber was coupled to a blue LED module (PlexBright blue LED module, 470 nm, Plexon, Inc) and intensity was controlled through a current generator (Plexon, LED Driver LD-1, Plexon, Inc). An output current of 50 mA was sufficient to produce a light power ranging between 0.2 ± 0.06 mW (irradiance = 1.72 mW / mm²) at the outlet of the implanted cannula. Light power was measured ex vivo after the end of the sensory testing, using a fiber optic power meters with internal sensor (Thorlabs, Inc). The sensitivity to heat and cold was tested using the Hargreaves and Cold Plantar assay, respectively, before stimulation (baseline) and during stimulation (20 Hz burst for 1s, every 10 s). Both tests were run with a 1-day interval in between.

Slice preparation, optogenetics and whole cell recording
Spinal cord slices were prepared for electrophysiology experiments in a similar manner to previous studies 11. Mice were decapitated while under isoflurane anesthesia, and the vertebral column was rapidly dissected and placed in oxygenated ice-cold dissection solution (containing in mM: 65 NaCl, 105 sucrose, 2.5 KCl, 1.25 NaH PO , 25 NaHCO , 25 glucose, 0.5 CaCl , 7 MgCl ). The back of the animal was pinned to the bottom of a Sylgard-coated plate with the ventral surface facing up, and a laminectomy was performed to expose the spinal cord. The spinal cord was then carefully removed from the vertebral column and the lumbar and cervical enlargements were isolated. The ventral side of each spinal cord enlargement was then glued to an agar block, mounted in a slicing chamber and installed in a vibrating blade microtome (D.S.K microslicer DTK1000). Transverse slices of 300 - 350 µm thickness were taken and transferred to a holding chamber filled with oxygenated artificial cerebrospinal fluid (aCSF) (containing in mM: 120 NaCl, 26 NaHCO , 1.25 NaH PO , 2.5 KCl, 5 HEPES, 14.6 glucose, 2 CaCl , 1 MgCl ; pH 7.35 - 7.40, 305 - 315 mOsm) warmed to 34°C. Slices were left to recover for at least 30 min before recordings were taken.

For the preparation of hindbrain slices, brains were quickly removed following euthanasia of the animal. The brain was placed in ice cold dissection solution, and the hindbrain was isolated by removing the forebrain and cerebellum. The hindbrain was glued to an agar block and transverse sections were cut at 250 µm, which were held in an incubation chamber filled with warm aCSF (34C) until recordings were taken.

Targeted whole-cell recordings were taken at room temperature using a HEKA EPC10 amplifier with Patchmaster software (HEKA Elektronik) at a sampling frequency of 20 kHz. For optogenetic experiments, cells in the spinal dorsal horn (laminae I-III) were randomly targeted using DIC optics. A Cs-based internal solution was used (containing in mM: 120 CsCl, 10 HEPES, 0.05 EGTA, 2 MgCl , 2 Mg-ATP, 0.1 Na-GTP, 5 QX-314) and recordings were taken in voltage clamp mode, with blue light stimuli (4 ms, 473 nm) delivered through the objective lens at a frequency of 0.1 Hz from a monochromator (TILL photonics). To confirm the inhibitory nature of light evoked synaptic events, bicuculline (20 µM) and strychnine (0.5 µM) were bath applied to the slice during recording.

For targeted recordings of RVM projection neurons, the spinal cords of vGATcre animals were injected with AAV2retro.flex.tdTOM or AAV2retro.flex.eGFP one week before the preparation of hindbrain slices. Labeled cells were visualized by epifluorescence illumination using a monochromator and were targeted for whole-cell recording. Recordings were taken in current clamp mode using a potassium gluconate internal solution (containing in mM, 130 K-Gluconate, 5 NaCl, 1 EGTA, 10 HEPES, 5 Mg-ATP, 0.5 Na-GTP, 2 biocytin) to determine spontaneous activity. Following recording, the recording electrode was slowly retracted, and the slice was fixed overnight at 4° in 4% PFA. Fixed hindbrain slices were processed to reveal biocytin-labeled cells and immunoreacted to confirm their tdTOM or eGFP content.

Access resistance was monitored throughout each experiment, and traces from each experiment were analyzed further if the access resistance changed < 30% during the recording. For light-evoked responses displayed in figure 4, averaged traces from 10 consecutive stimuli are shown in black, and individual traces used to generate the averaged trace are shown in grey. Blue lines indicate the time of blue-light stimulation, and all traces are aligned to this.

Spinal cord in vivo electrophysiology
Extracellular single-unit recordings were made from neurons in the superficial dorsal horn (100 – 300 μm from the dorsal surface) as described previously 78. In brief, mice were anesthetized with 2% isoflurane and maintained at 37°C body temperature. After T13 - L2 laminectomy, the lumbar segments were exposed, and the animal was mounted in a stereotaxic frame (David Kopf Instruments). A glass insulated carbon filament electrode (4 - 6 MΩ) was used for recording. Neurons were identified by monitoring background activity and responses to innocuous and noxious search stimuli. Neurons were selected that responded more strongly to noxious than innocuous stimuli, corresponding to the WDR type. For optogenetic activation of the superficial dorsal horn axon terminals of vGAT RVMSC neurons, AAV9.hEF1a.Dio.ChR2 was injected stereotaxically into the RVM of 6-week old vGATcre mice (control mice were injected with rAAV9.EF1a.Dio.mcherry). Recordings were made 3 - 4 weeks after vectors injections. Neuronal activity (spikes/s) was measured for 1 - 3 min in the absence of intentional stimulation (background activity) and during innocuous and noxious mechanical test stimulation. To assess the impact of vGAT RVMSC neuron activity on the responses of the recorded neuron, dorsal horn axon terminals of vGAT RVMSC neurons were stimulated with blue light (473 nm, 2 min, 20 Hz, 5 - 10 mW) using an optical fiber placed close to the recording electrode. Background and evoked activity of neurons was recorded before (baseline), during (“ON”), and after (“OFF”) blue light stimulation.

Statistical tests
Normal distribution was tested for all data sets using the Shapiro-Wilk test. If the data set followed a normal distribution, unpaired or paired t tests, or ANOVA were used. If the data set did not pass, non-parametric tests were used to assess statistical significance. p < 0.05 was considered statistically significant.

Data collection, storage, and presentation
Data were analyzed and presented using GraphPad Prism 8. Figures were arranged in Affinity Designer 2.

Material and Data availability
All unique materials are available from the authors. All quantitative data generated in this study are provided in the Source Data file. Raw data acquired in these experiments has been uploaded to https://doi.org/10.5281/zenodo.18983374 and is available for download.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this report, authors using optogenetic methods to re-investigate descending inhibition of pain from the RVM to the spina cord. Although it is great to see similar findings as previous reports (see Reviews from Sandkuhler, Gebhart et al), the study lacks of novel mechanisms for such modulation. Only behavioral responses were used for the evaluation of nociceptive responses, additional electrophysiological recordings from dorsal horn neurons combined with optogenetic stimulation may strength the argument.\nMajor:\n1. Authors failed to acknowledge previous reports of biphasic (facilitatory and inhibitory) descending transmission from this regions (see Zhuo and Gebbhart for spinal electrophysiology; Porreca for chronic pain). I wonder it may be caused by the limitation of techniques. How intensity-dependent stimulation may play out in light stimulation? does virus affect the healthy status of RVM neurons?\n2. Authors need to mention ON- off- cells reported by Fields et al for RVM descending modulation. I wonder if they are belonged to the types of cells in the RVM.\n3. Additional recordings from dorsal horn neurons, including their responses to peripheral thermal/mechanical stimuli are needed to confirm the inhibition at sensory transmission. Some control experiments from vspinal motor neurons would be helpful too.\n4. Authors need to cite old literature from Wills, Gebhart, Sandkuhler labs that consistently demonstrate that descending inhibitory modulation from the RVM are bilateral to spinal cord dorsal horn neurons. It is good to have virus methods to confirm this, however, it alone is not novel.", "reviewer_2": "The manuscript by Ganley et al uses viral genetic tools for tracing and behavioral assessment of brainstem neurons that influence nociceptive thresholds, mechanical and/or thermal in mice.\n1. There have been several studies of how the RVM impacts nociception, and thus the authors should put into context better which findings reported in this manuscript are novel to the field and why. It should be very clear to the broad audience of Nature Communications what information was lacking and what is now provided. There are only vague sentences in intro and discussion. Both the intro and discussion need to be re-written for a broader audience to include more in-depth explanations.\n2. The authors show that back-labeling bilaterally from the lumbar dorsal horn using an intersectional strategy with VGAT Cre and retro AAV captures GABAergic neurons in the RVM including in the NRM, Gi, LPGi. Using two different colored reporters for the back-labeling of ipsi and contra, the authors show that a significant proportion of RVM neurons project bilaterally. This is true whether both injections are at lumbar area or one is lumbar and the other cervical – hence widespread innervation along the cord by single RVM neurons.\n3. The study also highlights that these GABAergic RVM neurons also project to supraspinal areas, which is a confound for the study since the spinal projections are not specifically manipulated. The authors should provide a more compelling and detailed explanation for why these circuits are not responsible for their observations. It is dismissed only with a brief and nondescript sentence in the discussion. If a compelling explanation cannot be provided, experimental evidence that supports a dependence specifically on the spinal projections will be needed.\n4. hM3Dq -mediated activation of GABAergic RVM neurons from lumbar ipsilateral injection. Impact on von Frey, heat, cold but not pinprick or brush. Should discuss why not pinprick or brush. With respect to brush, inhibition of the neurons with TetLC impacts brush.\n5. hM3Dq -mediated activation of the GABAergic RVM neurons labeled from lumbar unilateral injection impacts the Hargreaves (heat sensitivity) across all four paws equally but the effect on cold sensitivity occurs most profoundly at the ipsi hind paw – this should be discussed.\n6. TetLC mediated inhibition of GABAergic RVM neurons labeled from lumbar unilateral injection increases sensitivity to brush, von Frey but no impact on heat or cold. There is some concern that the expression of TetLC is not as widespread or robust (indirectly looking at GFP) compared to the hM3Dq indirectly looking at mCherry. Also, no image of the dorsal horn GFP expression in the TetLC mice. An experiment that demonstrates the TetLC is working well is needed here. For example, co-inject the TetLC and the hM3Dq and show that the impact of CNO-mediated activation of the RVM neurons on heat and cold is blocked.\n7. Taken together, the results as shown indicate activating RVM GABA neurons can suppress heat, cold, and von Frey and inhibiting RVM GABA neurons can increase brush and von Frey. In this sense, only von Frey sensitivity seems to be bidirectionally modulated.\n8. Slice recordings of the RVM GABA neurons show some are tonically active. The discussion of these neurons in relation to the behavior could be explained in more detail- provide the logic or rationale for why tonically active neurons are modulating mechanical sensitivity in a more localized fashion (i.e. inhibiting them only impacted mechanical and only at hind paw).\n9. The impact of hM3Dq activation of GABAergic RVM neurons on CFA von Frey and CCI von Frey were different. CFA von Frey showed much greater recovery -going over baseline levels. CCI did not recover to baseline. Heat sensitivity with CFA recovered. The impact between CFA and CCI is therefore not the same and CCI has inflammatory and neuropathic components so perhaps CCI recovery reflects an impact on inflammatory component only.\n10. Show Fos induction in GABA RVM neurons due to injection of capsaicin in forepaw – shown in Fig. 7. In Figure 8, FosTrap2 mice are used which allows excitatory and inhibitory neurons in RVM to be captured with cap forepaw injection. With n=2, most neurons are GABAergic 80 vs 20%. Captured with hM3Dq. Do these neurons project to other brain areas or only to the spinal cord? Number of neurons is not reported but looks sparse and limited to LPGi. Reactivation of these neurons increases heat and cold thresholds but has no impact on von Frey or brush.\nThe authors should make it clear if the identification of LPGi neurons as mediators of CPM or DNIC is novel. The authors in the discussion mention it as a feed-forward circuit but do not explain why it is feed forward rather than feedback. The paragraph on DNIC with respect to the identified circuit and the method of activation is not clear. For example, the authors mention it is activated by noxious stimuli at a distant site but what does distant site mean?\nOther comments\nFigure 1A – missing v for VGLUT2 on graph\nFigure 2C left side- the labeling of brain regions in the slices is unclear.\nFigure 3 should say that behavior is measured at ipsilateral hind paw in figure legend.\nFigure 4B- the way the authors show the impact of strychnine and bicuculine on the ChR2 evoked currents is unclear. I assume the currents in gray are polysynaptic and that the flat also gray line under the currents is what was observed in the presence of antagonists but this is normally shown above the currents lining up with the light activation (blue line).\nFigure 4F- the labels are cutoff.\nTypo Page 10 line 3.", "reviewer_3": "The manuscript by Ganley et al is an intersectional study investigating he basis of RVM mediated inhibition of superficial dorsal horn (DH) physiology and thus descending inhibition of nociception. This is an important area of study in pain research as it is one of key cornerstones of clinically utilised analgesia, yet the fundamental properties of this process is not understood. The MS is well written and in large part the experiments are well designed and well presented. I do have a few concerns at this stage which I will outline below.\nMy first concern is the use of male mice exclusively in this study. There is a growing literature that pain processing differs between the sexes and that this is the case in PAG mediated analgesia, much of which relies on spinal projections from the RVM. The authors fail to investigate whether sex has an impact on their findings. Ideally this would be performed but would require considerable time, effort and expense. At a minimum some discussion of sex-based differences is required and an acknowledgement that the findings of this study are may not be applicable to females.\nNow to the second major concern. Was the C-FOS used as a proxy for identification of neuronal activity? Had the authors thought of performing RVM recordings to assess whether ON, OFF or neutral cells were impacted by the CNO treatment? This point then reflects my main concern with many of the outcomes from slice-based electrophysiology. Surely an in vivo approach to spinal and RVM recordings would have enabled many of the same data to be produced and allowed these to me made in an intact physiological context? Further it would have allowed for a clarification/classification of the neurons in the RVM manipulated and therefore permitted comparison from physiological and pharmacological studies in the literature. This is a key deficit in the field that the neurochemistry of physiological defined RVM neurons which respond to noxious stimuli is less than completely understood.\nMany of the points raised by the authors rely on extrapolations rather than direct measures. In terms of DNIC (and clearly not CPM) mechanistic insight is gained via whole animal, in vivo, electrophysiological and behavioural measures. Whether DNIC can be perturbed by activation/inhibition of RVM neurons requires an intact prep. Is it possible for a small set of studies to be conducted to confirm these ex vivo data from this study? This is especially important for the studies reflected in figure 4 which investigate functional synapses, yet use slices with isolated spinal cord in addition to the whole animal behaviour.\nPanel 3B and C seems a little out of focus (this may be my PDF) the lumbar injection site images are a little too low powered to enable a good inspection of superficial DH.\nThe title for the legend of figure 6 implies only a reversal of inflammatory pain yet CCI mediated hyperalgesia is partially reversed too, a change would help the reader.\nThe methods are unclear in some elements of experimental design. How were group sizes (power) calculated? How were animals assigned to treatment groups? Blinding is mentioned with regards to CNO treatment but there are other interventions in these studies in which experimenters should have been blind to- were they? There are a great many studies described in this study, some more detail on statistical treatment and the justification for those tests is required. For example was normality testing of all data sets performed?"}, "author_response": "Point-by-point reply\nReviewer 1:\nIn this report, authors using optogenetic methods to re-investigate descending inhibition of pain from the\nRVM to the spina cord. Although it is great to see similar findings as previous reports (see Reviews from\nSandkuhler, Gebhart et al), the study lacks of novel mechanisms for such modulation. Only behavioral\nresponses were used for the evaluation of nociceptive responses, additional electrophysiological\nrecordings from dorsal horn neurons combined with optogenetic stimulation may strength the argument.\nMajor:\n1. Authors failed to acknowledge previous reports of biphasic (facilitatory and inhibitory) descending\ntransmission from this regions (see Zhuo and Gebbhart for spinal electrophysiology; Porreca for chronic\npain). I wonder it may be caused by the limitation of techniques. How intensity-dependent stimulation may\nplay out in light stimulation? does virus affect the healthy status of RVM neurons?\nWe thank the reviewer for this advice. We now discuss previous work by the Gebhart and Porreca labs on\nbiphasic descending pain control by the RVM (p.10). In our study, we have used adenoassociated viruses\n(AAVs) for gene delivery. AAVs are well tolerated by neurons and even considered for gene therapy in\nhuman patients (e.g. PMID 40076831). Our functional (electrophysiological and chemogenetics) and\nmorphological analyses of AAV transfected neurons failed to reveal any signs of neuronal toxicity. We\nattribute the selective inhibitory effect of chemogenetic activation of vGAT RVMSC neurons to the specificity\nof our approach rather than to neurotoxic effects of AAVs.\n2. Authors need to mention ON- off- cells reported by Fields et al for RVM descending modulation. I wonder\nif they are belonged to the types of cells in the RVM.\nWe thank the reviewer also for this comment. We discuss ON, OFF and NEURTAL RVM cells in the revised\nmanuscript (p.10). The descending vGAT RVM neurons studied in the present manuscript share many\ncharacteristics with OFF cells.\n3. Additional recordings from dorsal horn neurons, including their responses to peripheral\nthermal/mechanical stimuli are needed to confirm the inhibition at sensory transmission. Some control\nexperiments from vspinal motor neurons would be helpful too.\nWe agree with the reviewer that in vivo recordings from dorsal horn WDR neurons would strengthen our\nstudy. Therefore, we initiated a collaboration with the Neugebauer lab at the Texas Tech University Health\nScience Center. Consistent with the effects of vGAT RVMSC neurons occurring in the spinal cord, the new\nexperiments show that optogenetic stimulation of descending vGAT RVMSC virtually abolishes the\nresponses of dorsal horn WDR neurons to noxious stimulation (revised figure 3 and extended data figure 3\nand p.6).\n4. Authors need to cite old literature from Wills, Gebhart, Sandkuhler labs that consistently demonstrate\nthat descending inhibitory modulation from the RVM are bilateral to spinal cord dorsal horn neurons. It is\ngood to have virus methods to confirm this, however, it alone is not novel.\nPrevious work e.g. by the Sandkuhler lab from 1995 and 1996 (PMIDs 7675181and 8931107) used kainate\ninjections into the ventromedial medulla (targeted onto the nucleus raphe magnus (NRM) to activated RVM\nneurons. Subsequent activation of spinal neurons was detected as c-fos expression. c-fos induction\noccurred bilateral and along the rostrocaudal axis. Although very informative at that time, with the\ntechniques available it was not possible to (a) selectively activate descending RVM neuron, (b) restrict the\nactivation to a certain neurochemically defined neuron type, and (c) to detect inhibition of spinal cord\nneurons. Our new study therefore significantly advances previous knowledge. Our new tools allowed us to\nspecifically activate genetically defined subsets of RVM neurons, to restrict activation to spinally\nprojecting neurons and to assess the effects of not only excitation but also inhibition. Our intersectional\n- 1 -\napproaches unequivocally demonstrate for the first time that individual vGAT RVMSC neurons traced from\nthe lumbar spinal cord innervate both sides of the spinal cord along its rostrocaudal axis.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Ganley et al uses viral genetic tools for tracing and behavioral assessment of brainstem\nneurons that influence nociceptive thresholds, mechanical and/or thermal in mice.\n1. There have been several studies of how the RVM impacts nociception, and thus the authors should put\ninto context better which findings reported in this manuscript are novel to the field and why. It should be\nvery clear to the broad audience of Nature Communications what information was lacking and what is now\nprovided. There are only vague sentences in intro and discussion. Both the intro and discussion need to be\nre- written for a broader audience to include more in-depth explanations.\nWe have re-written major parts of the discussion and now put our finding in the context of previous work\non descending pain modulation by the RVM and on earlier studies addressing DNIC and CPM (see also our\nresponses to reviewer 1, points 1 and 4). We state more clearly what was already known and what our\nstudy adds through the use of state-of-the-art neuroscience tools, such as viral tracing, and intersectional\ngenetics based chemogenetics.\n2. The authors show that back-labeling bilaterally from the lumbar dorsal horn using an intersectional\nstrategy with VGAT Cre and retro AAV captures GABAergic neurons in the RVM including in the NRM, Gi,\nLPGi. Using two different colored reporters for the back- labeling of ipsi and contra, the authors show that\na significant proportion of RVM neurons project bilaterally. This is true whether both injections are at\nlumbar area or one is lumbar and the other cervical – hence widespread innervation along the cord by\nsingle RVM neurons.\nWe thank the reviewer for this encouraging statement.\n3. The study also highlights that these GABAergic RVM neurons also project to supraspinal areas, which is\na confound for the study since the spinal projections are not specifically manipulated. The authors should\nprovide a more compelling and detailed explanation for why these circuits are not responsible for their\nobservations. It is dismissed only with a brief and nondescript sentence in the discussion. If a compelling\nexplanation cannot be provided, experimental evidence that supports a dependence specifically on the\nspinal projections will be needed.\nThe reviewer points to an important aspect of our study. We agree that the work cited in the previous\nversion of our manuscript may not be considered conclusive enough to exclude an antinociceptive effect\nvia RVM input occurring through supraspinal sites. Therefore, in our revised manuscript we added in vivo\nelectrophysiological experiments in collaboration with the Neugebauer lab at the Texas Tech University\nHealth Science Center. These experiments show that optogenetic stimulation of the spinal terminals of\nvGAT RVMSC neurons is sufficient to virtually block activation of dorsal horn wide dynamic range neurons\nby nociceptive input.\n4. hM3Dq -mediated activation of GABAergic RVM neurons from lumbar ipsilateral injection. Impact on von\nFrey, heat, cold but not pinprick or brush. Should discuss why not pinprick or brush. With respect to brush,\ninhibition of the neurons with TetLC impacts brush.\nThe differential impact of chemogenetic activation and tetanus toxin-mediated silencing on thermal\nversus mechanical sensitivity may suggest the presence of different populations of vGAT RVMSC neurons\ntargeting different dorsal horn neuron populations as discussed in the manuscript (p.10, last paragraph).\nWithout a detailed knowledge of the spinal target neurons of the vGAT RVMSC neuron populations, we can\nonly speculate about the underlying mechanisms.\n- 2 -\n5. hM3Dq -mediated activation of the GABAergic RVM neurons labeled from lumbar unilateral injection\nimpacts the Hargreaves (heat sensitivity) across all four paws equally but the effect on cold sensitivity\noccurs most profoundly at the ipsi hind paw – this should be discussed.\nWe agree with the reviewer that this is an interesting finding. As discussed above, at present, we could only\nspeculate about the underlying circuits.\n6. TetLC mediated inhibition of GABAergic RVM neurons labeled from lumbar unilateral injection increases\nsensitivity to brush, von Frey but no impact on heat or cold. There is some concern that the expression of\nTetLC is not as widespread or robust (indirectly looking at GFP) compared to the hM3Dq indirectly looking\nat mCherry. Also, no image of the dorsal horn GFP expression in the TetLC mice. An experiment that\ndemonstrates the TetLC is working well is needed here. For example, co-inject the TetLC and the hM3Dq\nand show that the impact of CNO-mediated activation of the RVM neurons on heat and cold is blocked.\nTetanus toxin light chain (TetLC)-mediated silencing is usually much more potent than chemogenetic\napproaches. We now provide a high-resolution image of the of the injected area (Fig.5C) which illustrates\nthat virtually all retrogradely labeled (magenta) neurons also express TetLC (green), with basically no\nmagenta-only neurons visible in the panel of Fig. 5C.\n7. Taken together, the results as shown indicate activating RVM GABA neurons can suppress heat, cold,\nand von Frey and inhibiting RVM GABA neurons can increase brush and von Frey. In this sense, only von\nFrey sensitivity seems to be bidirectionally modulated.\nWe agree with the reviewer.\n8. Slice recordings of the RVM GABA neurons show some are tonically active. The discussion of these\nneurons in relation to the behavior could be explained in more detail- provide the logic or rationale for why\ntonically active neurons are modulating mechanical sensitivity in a more localized fashion (i.e. inhibiting\nthem only impacted mechanical and only at hind paw).\nWe thank the reviewer for comment. We discuss a possible heterogeneity of vGAT RVMSC neurons on page\n10.\n9. The impact of hM3Dq activation of GABAergic RVM neurons on CFA von Frey and CCI von Frey were\ndifferent. CFA von Frey showed much greater recovery -going over baseline levels. CCI did not recover to\nbaseline. Heat sensitivity with CFA recovered. The impact between CFA and CCI is therefore not the same\nand CCI has inflammatory and neuropathic components so perhaps CCI recovery reflects an impact on\ninflammatory component only.\nThe contribution of inflammation to various nerve injury models in an interesting question. It is well\nestablished that macrophages and cytokines contribute to nerve injury-mediated sensitization (e.g., PMID\n20003309; PMID 27387067). In our previous experiments, we have shown that a pathway that is critically\ninvolved in inflammatory hyperalgesia does not contribute to sensitization in the CCI model (PMID\n16846696). We feel that the present wording is appropriate, as, in the abstract, we speak of chronic pain\nstates, and, in the results section, we specifically define the model used in our experiments.\n10. Show Fos induction in GABA RVM neurons due to injection of capsaicin in forepaw – shown in Fig. 7. In\nFigure 8, FosTrap2 mice are used which allows excitatory and inhibitory neurons in RVM to be captured\nwith cap forepaw injection. With n=2, most neurons are GABAergic 80 vs 20%. Captured with hM3Dq. Do\nthese neurons project to other brain areas or only to the spinal cord? Number of neurons is not reported\nbut looks sparse and limited to LPGi. Reactivation of these neurons increases heat and cold thresholds\nbut has no impact on von Frey or brush.\n- 3 -\nThe TRAP captured neurons project to similar supraspinal sites (lateral parabrachial nucleus and PAG) as\nthe neurons labeled from the spinal cord.\nPAG\nLbp\nLPGi\nThe authors should make it clear if the identification of LPGi neurons as mediators of CPM or DNIC is novel.\nWhile the RVM (or more specifically the NRM) has already previously been proposed as a CNS area\ninvolved in DNIC, we are not aware of studies that specifically identify a contribution of neurons in the\nLPGi.\nThe authors in the discussion mention it as a feed-forward circuit but do not explain why it is feed forward rather than feedback.\nWe have removed this statement, as, depending on the viewpoint, it may also be considered feedback.\nThe paragraph on DNIC with respect to the identified circuit and the method of activation is not clear.\nWe have re-written the discussion on DNIC and CPM (p.11) and hope that it is now clearer.\nFor example, the authors mention it is activated by noxious stimuli at a distant site but what does distant\nsite mean?\nWe use the term distant to explain that the test stimulus and the conditioning stimulus were applied on\ndifferent sides (left versus right) and at different levels (e.g., forepaw versus hindpaw). We hope that this is\nmade clear in the revised manuscript.\nPrevious work has suggested an involvement of OFF cells in the RVM as mediators of DNIC nucleus raphe\nmagnus\nWe thank the reviewer for this comment and quote the paper by Chebbi and colleagues (PMID 24681000).\nOther comments\nFigure 1A – missing v for VGLUT2\nhas been corrected\non graph Figure 2C left side- the labeling of brain regions in the slices is unclear.\nhas been corrected\nFigure 3 should say that behavior is measured at ipsilateral hind paw in figure legend.\nhas been corrected.\nFigure 4B- the way the authors show the impact of strychnine and bicuculine on the ChR2 evoked currents\nis unclear. I assume the currents in gray are polysynaptic and that the flat also gray line under the currents\nis what was observed in the presence of antagonists but this is normally shown above the currents lining\nup with the light activation (blue line).\nwe have re-arranged Fig. 4B\n- 4 -\nFigure 4F- the labels are cutoff. Typo Page 10 line 3.\nhas been corrected.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Ganley et al is an intersectional study investigating he basis of RVM mediated inhibition\nof superficial dorsal horn (DH) physiology and thus descending inhibition of nociception. This is an\nimportant area of study in pain research as it is one of key cornerstones of clinically utilised analgesia, yet\nthe fundamental properties of this process is not understood. The MS is well written and in large part the\nexperiments are well designed and well presented. I do have a few concerns at this stage which I will\noutline below.\nMy first concern is the use of male mice exclusively in this study. There is a growing literature that pain\nprocessing differs between the sexes and that this is the case in PAG mediated analgesia, much of which\nrelies on spinal projections from the RVM. The authors fail to investigate whether sex has an impact on\ntheir findings. Ideally this would be performed but would require considerable time, effort and expense. At\na minimum some discussion of sex-based differences is required and an acknowledgement that the\nfindings of this study are may not be applicable to females.\nIn the present study we have used mice of both sexes for most experiments. In the revised figure we\nindicate the sex of the mice using different symbols (filled versus open circles for male and female mice,\nrespectively). Some of the morphological experiments and the in vivo electrophysiology were done in male\nmice only. This is also indicated in the figures.\nNow to the second major concern. Was the C-FOS used as a proxy for identification of neuronal activity?\nHad the authors thought of performing RVM recordings to assess whether ON, OFF or neutral cells were\nimpacted by the CNO treatment?\nc-fos labeling was used as a marker of neuronal activation. We agree that in vivo recordings of RVM\nneurons ideally performed in behaving mice would provide more information, especially because of the\ntemporal resolution, which is completely lacking in case of c-fos stainings. However, such recordings are\nextremely challenging. They require surgery, implantations of electrodes, optical fibers or lenses. Given\nthat he LPGi is a very small structure at a delicate site of the brain stem, it is likely that such manipulations\nwould have interfered with the integrity of the circuit. We have however performed in vivo recordings from\nspinal wide dynamic range neurons in the spinal cord (Fig. 3 E,F and page 6).\nThis point then reflects my main concern with many of the outcomes from slice-based electrophysiology.\nSurely an in vivo approach to spinal and RVM recordings would have enabled many of the same data to be\nproduced and allowed these to me made in an intact physiological context? Further it would have allowed\nfor a clarification/classification of the neurons in the RVM manipulated and therefore permitted\ncomparison from physiological and pharmacological studies in the literature. This is a key deficit in the\nfield that the neurochemistry of physiological defined RVM neurons which respond to noxious stimuli is\nless than completely understood.\nWe certainly agree with reviewer that in vivo recordings from descending RVM neurons would add to our\nstudy. However, as pointed out above, we cannot currently overcome the many challenging technical\nrequirements of such experiments. We discuss some work on the neurochemical characteristics of ON,\nOFF and NEURTRAL RVM cells (p. 10).\nMany of the points raised by the authors rely on extrapolations rather than direct measures. In terms of\nDNIC (and clearly not CPM) mechanistic insight is gained via whole animal, in vivo, electrophysiological\nand behavioural measures. Whether DNIC can be perturbed by activation/inhibition of RVM neurons\nrequires an intact prep. Is it possible for a small set of studies to be conducted to confirm these ex vivo\n- 5 -\ndata from this study? This is especially important for the studies reflected in figure 4 which investigate\nfunctional synapses, yet use slices with isolated spinal cord in addition to the whole animal behaviour.\nWe appreciate the reviewer’s suggestion and collaborated with the Neugebauer lab at the Texas Tech\nUniversity Health Science Center to perform electrophysiological recordings from dorsal horn neurons in\nan intact preparation. We optogenetically stimulated the spinal terminals of vGAT RVMSC neurons and\nmonitored the impact of this stimulation on the activity of dorsal horn wide dynamic range neurons evoked\nby noxious and innocuous stimulation of their receptive field. These experiments revealed that optogenetic\nstimulation virtually abolished stimulus evoked action potential firing in these neurons.\nPanel 3B and C seems a little out of focus (this may be my PDF) the lumbar injection site images are a little\ntoo low powered to enable a good inspection of superficial DH.\nWe now show the images a higher resolution.\nThe title for the legend of figure 6 implies only a reversal of inflammatory pain yet CCI mediated\nhyperalgesia is partially reversed too, a change would help the reader.\nHas been changed.\nThe methods are unclear in some elements of experimental design. How were group sizes (power)\ncalculated?\nAs our project would be classified as “exploratory”, we did not perform sample size calculations but rather\nestimated required group sizes from previously conducted similar experiments by our group.\nHow were animals assigned to treatment groups?\nWhenever possible, we used a random number generator and the identification numbers of the mice in our\nanimal registration system to attribute mice to the different treatment groups. This was not possible when\ndifferent genotypes were compared. In these cases, we tested all mice in a certain litter with the correct\ngenotype.\nBlinding is mentioned with regards to CNO treatment but there are other interventions in these studies in\nwhich experimenters should have been blind to- were they?\nIn all behavioral experiments, the experimenter was blind either to the genotype of the animals or to their\ntreatment.\nThere are a great many studies described in this study, some more detail on statistical treatment and the\njustification for those tests is required. For example was normality testing of all data sets performed?\nWe have tested all data sets for normal distribution using the Shapiro-Wilk test. When the data set did not\npass this test, a non-parametric test was used to analyze for statistical significance. We have also added\nmore details on the outcomes of the statistical tests.\n- 6 -"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors have performed some additional experiments to address my concern on spinal nociceptive transmission. However, these experiments were only performed in normal mice but not chronic pain ones. It is well known that spinal nociceptive transmission is significantly enhanced after periphery injuries. In previous studies, many investigators reported that such inhibition was diminished in animal models of chronic pain. Maybe it will be good for authors to repeat such experiments using classic electrical stimulation, in order to compare with optogenetic methods. Secondly, descending facilitation from RVM to spinal cord has been well documented, it seems to be missing using optogenetic methods. Again, authors may need to repeat previous studies using old methods.", "reviewer_2": "With a few exceptions below, the authors addressed the earlier concerns. The work makes a significant contribution to the field.\nComments:\n1. “…neuropathic diseases.” There is a typo but also it is unclear that neuropathic component of pain was affected as stated the earlier review. Could have been only inflammatory component.\n2. It is not conclusive as described to say that the presence of an effect in one location (shutting down WDR lamina I neurons) is causal if it is unclear whether another effect is simultaneously happening in another location (modulation of supraspinal circuit). The physiological effect on spinal cord has to be isolated so impact on the spinal cord can be monitored. Behavior has to be measured in this same condition.\n3. Related to this, the following statement is too vague. “Furthermore, previous work has 333 shown that inhibitory effects of RVM stimulation required intact dorsolateral funiculi, 334 indicating that descending projections to the spinal cord are required for these effects 39, 40, 41, 335 42.”\nI can look up the references but can the authors just state what the reported inhibitory effects are that require descending projections?"}, "author_response": "Point-by-point reply\nReviewer #1 (Remarks to the Author):\nAuthors have performed some additional experiments to address my concern on spinal\nnociceptive transmission. However, these experiments were only performed in normal\nmice but not chronic pain ones.\nWe have carefully discussed the possibility of adding additional in vivo WDR neuron\nrecordings from diseased mice, which were notably not request in the first round of\nreviews. We are not convinced that these experiments would substantially strengthen\nthe manuscript. Our existing in vivo WDR recordings already indicate that the\nantihyperalgesic effects observed in behavioral pain tests are associated with reduced\naction potential firing in dorsal horn WDR neurons. We discuss this in our revised\nmanuscript (p. 10 / l. 7-12). In addition, we have performed behavioral experiments in\nboth inflamed and neuropathic mice that clearly demonstrate antihyperalgesic effects\nfollowing activation of descending RVM vGAT neurons. Based on these data, we\ncurrently have no reason to expect fundamentally different outcomes in mice with\npathological pain states.\nIt is well known that spinal nociceptive transmission is significantly enhanced after\nperiphery injuries. In previous studies, many investigators reported that such inhibition\nwas diminished in animal models of chronic pain.\nWe fully accept the reviewer’s comments. We have ourselves contributed to the\nidentification of mechanism that enhance spinal nociception in inflammatory and\nneuropathic pain states.\nMaybe it will be good for authors to repeat such experiments using classic electrical\nstimulation, in order to compare with optogenetic methods.\nIt is not unexpected that experiments employing classical, non-specific electrical or\nchemical stimulation of all RVM neurons and genetics-based approaches that\nselectively recruit the spinally projecting inhibitory RVM neurons yield different results.\nWe feel that repeating these early experiments using non-specific methods would not\nbe meaningful when newer, more precise techniques are available. Instead, we will\nacknowledge this point in our Discussion (p. 10 / l. 36 – p. 11 / l. 2).\nSecondly, descending facilitation from RVM to spinal cord has been well documented, it\nseems to be missing using optogenetic methods. Again, authors may need to repeat\nprevious studies using old methods.\nWe agree with the reviewer that stimulation of the RVM can have opposing effects on\nspinal nociception. We now discuss the heterogeneity of responses in the revised\nmanuscript (p. 10 / l. 37 – p. 11, l. 3 and p. 11 / l. 29-30). Facilitating responses have in\nfact been reported also after optogenetic stimulation (François et al., Neuron 2017). As\nalready discussed, we attribute these differences to different subnuclei targeted in the\ndifferent studies (p. 11 / l. 20 – 28).\nReviewer #2 (Remarks to the Author):\nWith a few exceptions below, the authors addressed the earlier concerns. The work\nmakes a significant contribution to the field.\nComments:\n1. “…neuropathic diseases.” There is a typo but also it is unclear that neuropathic\ncomponent of pain was affected as stated the earlier review. Could have been only\ninflammatory component.\nWe have corrected the typo (thank you for the careful reading). We now discuss that the\nrelatively small antihyperalgesic effect of vGAT RVMSC neuron stimulation in the CCI\nmodel might potentially be due to its restriction to the inflammatory component of CCI\ninduced hyperalgesia (p.12/l.1 - 6). We also adapted the wording throughout the\nmanuscript and speak of “hyperalgesic disease states” or “pathological pain states”\ninstead of neuropathic pain to take this possibility into account.\n2. It is not conclusive as described to say that the presence of an effect in one location\n(shutting down WDR lamina I neurons) is causal if it is unclear whether another effect is\nsimultaneously happening in another location (modulation of supraspinal circuit). The\nphysiological effect on spinal cord has to be isolated so impact on the spinal cord can\nbe monitored. Behavior has to be measured in this same condition.\nWe appreciate the reviewer’s critique and have performed additional behavioral\nexperiments with local spinal chemogenetic or optogenetic stimulation (revised\nextended data figure 3 A-F, and p. 6/l. 5-15, and p. 10/l. 4-10).\n3. Related to this, the following statement is too vague. “Furthermore, previous work has\n333 shown that inhibitory effects of RVM stimulation required intact dorsolateral\nfuniculi, 334 indicating that descending projections to the spinal cord are required for\nthese effects 39, 40, 41, 335 42.”\nI can look up the references but can the authors just state what the reported inhibitory\neffects are that require descending projections?\nWe now explicitly mention the experiments described in the previous publication\n(p.10/l. 4-7)"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed remaining concerns."}, "author_response": "Point-by-point reply\nReviewer #2 (Remarks to the Author):\nThe authors have addressed remaining concerns.\nThank you for this positive comment."}]

Decision letter