Neurons in the caudal ventrolateral medulla mediate descending pain control

Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses. The caudal ventrolateral medulla was thought to be involved in pain control, but its pathway was unknown. Here, Gu et al. identify the molecular components of a caudal ventrolateral medulla–locus coeruleus–spinal cord pathway and show it has a role in counter-stimulus pain control.

Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive.
Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses. Painful stimuli are detected by peripheral nociceptive neurons, which subsequently transmit signals to higher brain centers to produce appropriate sensory percepts and to regulate physiological and behavioral responses. These responses can be modulated by descending circuits, which involves modification of the activity of spinal cord (SC) neurons [1][2][3] . These descending circuits often produce context-dependent effects, where modulation of pain likely has an adaptive advantage. For example, pain responses are suppressed during goal-directed activity such as feeding 4,5 , whereas injury can lead to facilitation of pain, which may be adaptive to allow optimal tissue recovery and regeneration 6 .
The modulation of pain is thought to be driven by several brain nuclei. About 50 years ago, it was shown that focal electrical stimulation of certain midbrain regions could elicit analgesia 7,8 . This led to studies that eventually showed that spinally projecting neurons in the ventral locus coeruleus (LC) and in the rostral ventral medulla (RVM) release norepinephrine and serotonin, respectively, in the SC, and these modulate the activity of nociceptive spinal circuits 9 . In addition to norepinephrine and serotonin, the LC and RVM release other transmitters that affect nociceptive responses, and other brain nuclei can also alter nociceptive signaling, including through corticospinal and bulbospinal pathways [10][11][12][13] . Regarding the LC, output from ventral areas evokes antinociceptive responses through SC projections, whereas output from dorsal areas induces pro-nociceptive responses through forebrain connections 14,15 . However, not all inputs to the LC that mediate pain control are well defined 16,17 .
In addition to the RVM and LC, the ventrolateral medulla (VLM) region is an important supraspinal center implicated in pain control 2 . Broadly defined, the VLM region consists of a number of subregions containing heterogeneous neurons in the ventrolateral quadrant of the medulla oblongata. Noxious stimulation leads to c-Fos expression in the VLM 18,19 . In addition, electrical stimulation of the VLM 20 , glutamate and GABA-agonist micro-injections into this region, elicit analgesia 21,22 , and lesioning of the VLM affects nociceptive responses 23 . These findings suggest that the broad VLM region regulates nociception, but the neurons mediating this process are unknown. In addition, follow-up studies suggest that there may be direct connections between the VLM and SC 24 , whereas other studies report indirect connections 13 . These opposing results likely arose because different classes of neurons were traced. In addition, the relationships of these neurons with the effects of VLM stimulation was not established. Altogether, previous studies suggest the VLM is a center for pain control, but the exact molecular identity and circuits involved remain undetermined.
Here we defined a population of noradrenergic neurons that are also glutamatergic in the VLM, which are activated by painful stimuli and demonstrate that these neurons are synaptically connected to the LC. Via the LC, VLM neurons produce SC-mediated control of nociceptive responses. Our findings reveal molecular detail for a Article https://doi.org/10.1038/s41593-023-01268-w stimuli. Consistent with our hypothesis, we observed increased intracellular calcium upon injection of capsaicin, challenge with noxious heat, and noxious mechanical pinch (Fig. 2b-f and Extended Data Fig. 2a). Calcium influx sharply increased when hot-plate temperatures started to reach the noxious heat threshold of mice and declined when temperature decreased (Fig. 2d,e). No change in fluorescence signal was detected in control mice injected with AAV2-DIO-GFP. In contrast to hot-plate stimulation, the mild noxious heat stimulation produced by the Hargreaves and cold plantar tests (transient localized subject-terminated thermal stimuli) as well as innocuous mechanical stimulation elicited minimal changes in calcium (Extended Data Fig. 2). Because the signal for these measurements comes from a small number of cVLM TH neurons, a potential limitation of this technique is that responses of neurons is below the threshold of this method. Nonetheless, our results show that cVLM TH neurons are preferentially activated by noxious stimuli.

Activation of cVLM TH neurons suppresses nociceptive responses
Our photometry experiments uncovered that cVLM TH neurons are sensors of noxious insults, suggesting that the cVLM might be involved in a pain pathway. To investigate how they might participate in pain signaling, we targeted expression of specific genes to these neurons using viral approaches. Specifically, we examined the behavioral consequences of unilateral stimulation of VLM TH neurons in TH-CreERT2 mice with designer receptors exclusively activated by designer drugs (DRE-ADD), which were expressed selectively and efficiently in these neurons ( Fig. 3a-d). This chemogenetic strategy to examine the cellular function should result in increased c-Fos staining in cVLM TH neurons. Figure 3e,f shows that, as expected, upon activation with clozapine N-oxide (CNO), there was a marked increase in numbers of c-Fos-positive TH neurons. We anticipated that if cVLM TH neurons are part of a nociceptive pathway, then their activation might elicit changes in withdrawal from painful stimuli. Indeed, activation of cVLM TH neurons evoked a profound suppression of responses to heat in Hargreaves tests ( Fig. 3g; see Extended Data Fig. 3a-c for controls), suggesting that cVLM TH cells may be part of a nociceptive modulatory system. Unexpectedly, although we performed unilateral cVLM stimulation, decreased sensitivity was induced in both ipsilateral and contralateral hind paws (diffuse inhibition) with both the right and left cVLM producing similar diffuse inhibition (Fig. 3h). In addition, further suggesting a role for these neurons in pain control, chemogenetic activation of cVLM TH neurons increased the latency for lick responses on a hot-plate assay, a behavior that requires supraspinal processing of nociceptive signals (Fig. 3i). By contrast, chemogenetic activation of cVLM TH neurons had no detectable effects on behavioral responses to itch, cooling and mechanical stimulation (including pinch) and additionally did not change core body temperature and motor coordination (Extended Data Fig. 4a-f).
A corollary of chemogenetic activation of cVLM TH neurons triggering decreased sensitivity to heat is that their inhibition, if they are tonically active in basal conditions, might elicit increased sensitivity. Indeed, DREADDi manipulation (Fig. 3j) diffusely reduced the latency of withdrawal responses in Hargreaves assays suggesting that they normally provide inhibitory tone in naïve conditions ( Fig. 3k; see Extended Data Fig. 3d,e for controls). Selective ablation of cVLM TH neurons, produced increased sensitivity to heat challenge (Fig. 3l). Similar to chemogenetic activation, altered response to heat stimuli was the only sensory modality where we detected changed responses to chemogenetic inhibition (Extended Data Fig. 5a-f).
While chemogenetics is a powerful technique to probe the function of neuronal ensembles, it offers low temporal precision. Therefore, we used optogenetics to examine the time course of cVLM-induced changes in behavioral responses and the time required, after cessation of stimulation, for deactivation (Fig. 4a). Similar to chemogenetic previously unappreciated circuit for pain control. Additionally, we provide evidence for a role of the VLM-LC pathway in counter-stimulusinduced analgesia.

Catecholaminergic VLM neurons are activated by noxious stimuli
Previously, it was reported that, after noxious stimulation, neurons in the caudal ventrolateral medulla (cVLM) express the marker of cell activation, c-Fos 18,19 . Using application of the potent pain-inducing agent capsaicin, we were able to replicate these findings showing a select group of neurons are activated by pain (Fig. 1a,b). Consistent with these neurons being specifically capsaicin activated, capsaicin administration in Trpv1-null animals failed to activate these neurons (Extended Data Fig. 1a,b). Intriguingly, despite using a unilateral stimulus, we found that increased c-Fos staining was bilateral, suggesting that the cVLM receives both ipsilateral and contralateral inputs (Fig. 1c). In addition, similar to the effects of capsaicin, intra-plantar injection of the pain-inducing agent ATP resulted in c-Fos expression in the cVLM (Fig. 1d). The molecular identity of these pain-activated neurons was unknown, and we wondered whether they might belong to a particular class. Based on their location in the VLM (bregma ~−7.7 to −7.9 mm), we reasoned that they might be catecholaminergic A1 neurons 25 . Therefore, we performed double-label immunohistochemistry with antibodies against c-Fos and tyrosine hydroxylase (TH). Results from these studies revealed that the majority of TH-positive VLM neurons stained for c-Fos ( Fig. 1a,b), and hereafter, we refer to these neurons as VLM TH neurons. To characterize cVLM TH neurons further, we performed multi-label in situ hybridization (ISH) to determine whether they are either excitatory or inhibitory and to test whether they express genes required for catecholamine synthesis. Figure 1e,f shows that almost all cVLM TH neurons express the glutamate transporter Vglut2, and we found that these neurons express genes required for production of norepinephrine (TH, DOPA decarboxylase (DDC) and dopamine β-hydroxylase (DBH)) but not epinephrine (phenylethanolamine N-methyltransferase (PNMT) was absent). In addition, the monoamine synaptic transporter Slc18a2 was present in these neurons (Extended Data Fig. 1c-e). To investigate whether SC projection neurons target cVLM TH neurons, we performed anterograde tracing studies with adeno-associated virus, serotype 1 (AAV1)-CAG-cre injected into the SC of Ai9 reporter mice 26,27 . We predicted that, if VLM TH neurons are postsynaptic to SC projection neurons, AAV1-cre would be transferred in the anterograde direction and lead to expression of tdTomato fluorescent reporter in VLM TH neurons. Although we observed tdTomato-positive cells adjacent to VLM TH neurons, almost no double-labeled neurons were observed (Fig. 1g,h). Despite not observing anterograde transfer of AAV1 to VLM TH neurons, we found labeled neurons in the RVM, the inferior olivary complex and the cortex using this technique (Extended Data Fig. 1f,g) 28,29 , confirming that this tracing technique can label the postsynaptic partners of SC projection neurons. Additionally, we used rabies virus tracing to investigate whether neurons in the SC are presynaptic to cVLM TH neurons. Consistent with anterograde studies, we did not find rabies-labeled neurons in the SC and found neurons surrounding the cVLM that may be presynaptic (Extended Data Fig. 1h,i). Taken together, our results show that noxious stimuli activate a relatively uniform class of excitatory noradrenergic neurons in the cVLM that are not postsynaptic to SC projection neurons.
Because the c-Fos expression technique does not readily allow analysis of many types of stimuli, we turned to in vivo fiber photometry to probe calcium responses in VLM TH neurons. TH-Cre mice were injected with AAV9-CAG-FLEX-GCaMP6s virus into the cVLM to monitor cellular activity of cVLM TH neurons in awake behaving mice (Fig. 2a). We hypothesized that because these neurons are activated by capsaicin, they might also be activated by other noxious stimuli. Therefore, we challenged animals with several noxious and innocuous  Fig. 1 | The cVLM TH , a brainstem nucleus activated by capsaicin. a, Immunostaining for c-Fos in the caudal medulla of mice where capsaicin was applied unilaterally to the hind paw revealed TH + neurons positive for c-Fos in the cVLM. b, Magnified view (boxed area in a) of the cVLM showed that almost all TH-labeled neurons were c-Fos positive after administration of capsaicin and few TH neurons were labeled for c-Fos after saline treatment. Scale bars, 500 µm (coronal sections) and 50 µm (magnified field images). c,d, Quantification of the percentage of c-Fos + TH + to TH + neurons after capsaicin (c) and ATP (d) treatment compared to PBS controls; P < 0.0001, n = 6 mice for capsaicin and P = 0.042, n = 3 mice for ATP, two-sided unpaired t-test. Data  Article https://doi.org/10.1038/s41593-023-01268-w stimulation, while expression of control mCherry had no effects, optogenetic activation was effective at reducing sensitivity to noxious heat (Fig. 4c,d; see Fig. 4b for controls). Interestingly, minutes after optogenetic stimulation had ended, there was residual suppression of responses to noxious heat showing that cVLM TH neurons activate a slowly desensitizing inhibitory circuit (Fig. 4d). Together these results establish that, in an apparent feed-forward inhibitory circuit, noxious stimuli activate cVLM TH neurons, which in turn participate in antinociception.

cVLM TH neurons are connected to a descending LC-SC pathway
To understand more about the potential mechanisms by which the cVLM TH ensemble might induce antinociception, we investigated the downstream targets of these cells. We identified their projection targets by injecting Cre-dependent AAVs expressing mCherry and synaptobrevin-GFP into the cVLM of TH-Cre mice to mark nerve fibers and the synapses of cVLM TH neurons, respectively (Fig. 5a). This approach revealed termination of axons in a number of brain nuclei. Of particular interest, we found strong labeling of VLM TH neuronal terminals in the LC (Fig. 5b-d and Extended Data Fig. 6a-f). Additionally, as reported previously, we uncovered projections to the periaqueductal gray (PAG), the paraventricular hypothalamus (PVN), the paraventricular thalamus (PVT), and the bed nucleus of the stria terminalis (BNST) 30,31 (Extended Data Fig. 7a-g). The LC is a brain nucleus well known to mediate analgesia through descending SC projections 15,32,33 , suggesting that it could be a downstream partner responsible for the antinociceptive effects of the cVLM. To determine whether the LC is functionally engaged by the cVLM, we used a number of complementary experimental approaches. First, using c-Fos expression as a metric for cell activation, we investigated whether activation of cVLM TH neurons is sufficient to activate LC neurons (Fig. 5e). For these experiments, we chemogenetically activated cVLM TH neurons in conjunction showed that capsaicin treatment significantly altered calcium responses compared to PBS-injected controls; P = 0.0025, n = 8 mice, two-sided paired t-test. d, Responses to heat challenge on a hot plate show intracellular calcium increased in cVLM TH neurons in the noxious temperature range, averaged responses ± s.e.m., n = 6 GCaMP6s mice (blue) and n = 5 GFP control mice (black traces). Quantification of the AUC for measurements are shown to the right and compared to those from mice injected with AAV2-DIO-GFP. GCaMP6s responses were significantly different from GFP responses; P = 0.0026, two-sided unpaired t-test. e, Heat map traces from six individual animals to heat challenge and for comparison changes in fluorescence observed in cVLM TH neurons expressing GFPexpressing control animals (lower five traces). f, Calcium responses to bulldog clamp on the tail, averaged responses ± s.e.m., n = 6 GCaMP6s mice (blue) and n = 5 GFP control mice (black traces). Quantification of the AUC for measurements are shown to the right and compared to those from mice injected with AAV2-DIO-GFP. GCaMP6s responses were significantly different from GFP responses; P = 0.031, two-sided unpaired t-test. In c, d and f, the box plots represent the 25th and 75th percentiles, whiskers represent the maximum and minimum values, and the horizontal line represents the mean. ΔF/F, change in fluorescence.
Article https://doi.org/10.1038/s41593-023-01268-w with labeling LC-SC-projecting neurons (by intraspinal injection of GFP-expressing AAV in the SC) and determined numbers of spinally projecting LC neurons positive for c-Fos. Using this approach, we observed robust activation of LC-SC neurons consistent with activation of LC neurons by the cVLM (Fig. 5f-h). Second, we assessed whether calcium responses in LC neurons can be evoked by the activation of the cVLM TH ensemble. We combined optogenetic stimulation of cVLM nerve terminals in the LC with calcium imaging of LC cells (Fig. 5I). Results from these experiments establish that the activation of LC neurons is tightly connected with stimulation of cVLM TH neurons (Fig. 5j,k). Thirdly, we examined whether activation of terminals of cVLM TH neurons projecting to the LC can modulate nociceptive responses (Fig. 5l). We injected AAV-hSyn-FLEX-Chrimson into the cVLM of TH-Cre mice and optogenetically stimulated cVLM TH nerve terminals in the LC. Figure 5m shows that the activation of cVLM TH fibers increased withdrawal latencies corroborating that the LC is an important route for cVLM-mediated inhibition of heat responses. We previously showed that activation of cVLM nerve fibers in the PVT induces increased food-seeking behavior 34 and we wondered whether activation of LC terminals would have a similar effect. Suggesting specificity, the optogenetic stimulation of the LC did not change feeding responses (Extended Data Fig. 8a,b). In addition, activation of PVT terminals did not alter sensitivity to heat in Hargreaves tests (Extended Data Fig. 8c) indicating distinct responses are driven by different cVLM outputs. These data strongly suggest that cVLM TH neurons activate the LC; however, they do not establish that neurons in these nuclei are synaptically connected. To investigate the coupling of cVLM TH neurons with the LC, we performed physiology experiments on coronal  h, Behavioral data for chemogenetic activation of cVLM TH neurons (CNO administration) comparing left versus right cVLM injection. There were no significant differences in behavior between L and R injected animals, n = 8 mice for each group, F = 0.3848, P = 0.765, one-way analysis of variance (ANOVA); data are the mean ± s.e.m. i, On the hot plate test (52 °C), the latency to lick was significantly altered in response to chemogenetic stimulation of cVLM TH neurons compared to saline controls, n = 8 mice, P = 0.024, two-sided paired t-test; data are the mean ± s.e.m. j, Schematic of the strategy used for chemogenetic inhibition of cVLM TH neurons. k, Chemogenetic inhibition with DREADDi caused a significant change in withdrawal latencies compared to saline controls, n = 11 mice, P < 0.0001 for L and P = 0.0002 for R hind paws, two-sided paired t-test; data are the mean ± s.e.m. l, Diphtheria toxin subunit A was expressed, after tamoxifen induction, in cVLM TH neurons of TH-CreER mice. Ablation of cVLM TH neurons caused a significant change in withdrawal latencies compared to saline controls, n = 11 mice, P < 0.0001 for L and, P = 0.0002 for R hind paws, two-sided paired t-test; data are the mean ± s.e.m. NS, not significant.  (Fig. 6b,c). These responses were blocked by tetrodotoxin (TTX) toxin application, an effect that could be partially reversed by co-application of 4-aminopyridine (4-AP) 35 . These findings support the existence of connectivity between cVLM TH and LC TH neurons. The connections between the cVLM and LC are likely complex and may contain both monosynaptic and polysynaptic connections. Consistent with the fact that spinally projecting LC TH neurons are mostly found in the ventral LC 15,36 , ventrally located cell bodies were preferentially activated by optogenetic stimulation (Fig. 6e). Additionally, we examined whether Calcium responses were affected by application of either beta-adrenergic or glutamate receptor antagonists. Interestingly, responses were attenuated by 2,3-dioxo-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX) plus 2-amino-5-phosphonovalerate (AP5), but not by propranolol (Extended Data Fig. 8f,g), suggesting that glutamate is the major transmitter at this particular synapse. Because the early phase of the response is driven by glutamate (as confirmed by NBQX and AP5), the transient response seen in Fig. 6b is not surprising. The result suggests that this synapse cannot sustain prolonged activation likely due to neurotransmitter/ vesicle depletion, which is observed in high-probability synapses when stimulated at high frequency (also see results in Fig. 5j). To provide further evidence for synaptic connection between cVLM TH and LC TH neurons, we performed retrograde rabies virus tracing studies (Fig. 6f,g). For these tracing studies, we injected the LC of TH-cre mice with Cre-dependent AAVs that express G-protein and TVA and then injected EnvA pseudotyped rabies virus (G-protein deleted; monosynaptic) into the LC 37 (Fig. 6f). As expected, this approach revealed a monosynaptic connection from LC TH neurons to cVLM TH neurons (Fig. 6g). Examination of the collaterals between the cVLM and the LC and the PVT revealed evidence for extensive collaterals of cVLM axons to these two nuclei (Extended Data Fig. 8d,e). Therefore, the results from the optogenetic experiments do not exclude the possibility that another (non-PVT) collateral may mediate the effect but, combined with the data in Figs. 6 and 7 (as well as Extended Data Fig. 8a-c), the findings strongly suggest that the pathway exploited for these responses occurs via the LC.

cVLM TH neuron-induced antinociception occurs in the SC
Our results suggest that the cVLM is part of an LC-to-SC pathway. Because a major neurotransmitter of LC neurons is norepinephrine and norepinephrine agonists are known to induce analgesia in the SC 32 , we wondered whether cVLM-induced antinociception might be attenuated by local SC delivery of alpha-2 adrenergic receptor antagonist. To test this postulate, we chemogenetically activated cVLM TH neurons and examined whether the injection of the alpha-2 adrenergic receptor antagonist yohimbine could block the cVLM TH chemogenetic-induced reduction in heat sensitivity (Fig. 7a). As anticipated for a descending SC noradrenergic pathway 32 , this treatment blocked cVLM chemogenetic-induced antinociception ( Fig. 7b) suggesting that a major descending pathway of the cVLM is via the SC neurotransmitter norepinephrine. In addition, as expected, we found that cVLM optogenetic-induced antinociception was almost completely inhibited by yohimbine treatment (Extended Data Fig. 9a,b).
To probe this postulate further we used an AAV-mediated CRISPR 38 genetic strategy to eliminate norepinephrine synthesis specifically in the LC TH neurons. We selectively expressed SaCas9 in LC neurons as well as a guide RNA, which targets the specific disruption of TH gene ( Fig. 7c) 38 . This manipulation resulted in more than an 80% reduction in cells expressing TH (Fig. 7d). As predicted, for norepinephrine being the major transmitter in SC in the cVLM-LC-SC circuit, loss of TH expression in LC neurons has a major effect on cVLM-dependent reduced sensitivity to heat (Fig. 7e). Furthermore, latency to lick responses in a hot plate assay was similarly attenuated by CRISPR-mediated gene disruption (Fig. 7f). Cumulatively, these studies establish that the LC and LC-derived norepinephrine are required for cVLM TH -dependent antinociception.
Given that norepinephrine is required for cVLM-induced antinociception, then cVLM TH -induced pro-nociception might be expected to be reduced by administration of an adrenergic receptor agonist (Fig. 7g). As predicted, clonidine (an αlpha-2 adrenergic receptor agonist) treatment almost completely alleviated chemogenetic (DREADDi)-mediated sensitization to heat (Fig. 7h). In contrast, pharmacological interventions of serotonin signaling 39,40 were ineffective at relieving cVLM-induced pro-nociception (Extended Data Fig. 9c,d). Together, these results demonstrate that the LC is both required and sufficient for cVLM-mediated control of heat antinociceptive responses providing confirmation of the cVLM-LC-SC circuit. data are the mean ± s.e.m. c, Optogenetic stimulation caused a significant change in withdrawal latencies in Hargreaves assays, compared to baseline, n = 7 mice, P = 0.014 for L and P = 0.0059 for R hind paws, two-sided paired t-test; data are the mean ± s.e.m. d, Time course for activation and deactivation of attenuated responses to heat challenge (Hargreaves test) following optogenetic stimulation; testing was performed before, during (blue line) and after optogenetic stimulation. There was a significant difference in withdrawal responses compared to baseline during stimulation and at 2 min after the end of optogenetic stimulation, P = 0.025 and P = 0.021 respectively; n = 7 mice, Dunnett's tests following one-way ANOVA.

The cVLM is required for counter-stimulus-induced analgesia
Results from our chemogenetic and optogenetic studies showed that the cVLM-LC-SC circuit can elicit a diffuse reduction in responses to heat. The characteristics of this circuit show some similarity to those reported for a number of descending pain pathways 1,2,41-44 . Some of these pathways have been shown to utilize norepinephrine and are reported to be controlled by supraspinal nuclei in the brainstem. State-dependent pain control, including counter-stimulus-induced analgesia, have been proposed to utilize these circuits. Therefore, we sought ways to test whether the cVLM can induce analgesia. We probed whether the activation of the cVLM circuit is sufficient to alleviate thermal allodynia (a pain state). We injected complete Freund's adjuvant (CFA) into a hind paw of mice to produce inflammatory pain and tested whether chemogenetic activation of cVLM TH neurons could reverse the resulting increased thermal sensitivity. Withdrawal latencies of the injured foot, after cVLM TH activation, were returned almost to baseline, demonstrating that cVLM TH neurons are sufficient to reverse thermal allodynia (Fig. 8a). These results agree with our finding that chemogenetic activation of the cVLM is sufficient to attenuate reactions to heat in hot plate tests (Figs. 3l and 7f). To further our understanding of potential roles for the cVLM, we next examined whether it might be involved in counter-stimulus-induced analgesia. Specifically, we examined whether calcium responses in cVLM TH neurons to noxious challenge are altered by a counter-stimulus and would act in a feed-forward circuit to inhibit input to itself. In these studies, we compared responses of cVLM TH neurons to a series of heat stimuli (three 55 °C ramps) before and after administration of a painful counter-stimulus (injection of 10 µg capsaicin into a forepaw; Fig. 8b,c). As expected, this counter-stimulus evoked delayed licking behavior to noxious heat (Extended Data Fig. 10b). In addition, as we previously observed, capsaicin treatment evoked a large increase in baseline calcium (Fig. 2b). This counter-stimulus, after subtraction of baseline, also resulted in significantly smaller calcium responses to heat challenge than those  k, Quantification of AUC responses showed that activation of Chrimsonexpressing fibers produced significantly different responses compared to mCherry-expressing fibers, n = 6 mice, P = 0.017, two-sided paired t-test; data are the mean ± s.e.m. l, Approach used to optogenetically activate LC-projecting cVLM TH neuronal fibers. m, Optogenetic stimulation of Chrimson in LC terminals caused a significant change in withdrawal latencies in L and R hind paws, in Hargreaves assays, compared to baseline, n = 8 mice, P = 0.0097 for L and P = 0.0196 for R hind paws, two-sided paired t-test; data are the mean ± s.e.m. n, Optogenetic stimulation of LC terminals expressing mCherry did not cause a significant change in withdrawal latency, n = 6 mice, P = 0.090 for L and P = 0.94 for R hind paws, two-sided paired t-test; data are the mean ± s.e.m. Scale bars, 500 µm (b), 50 µm (c) and 100 µm (g,h).
Article https://doi.org/10.1038/s41593-023-01268-w seen in naïve conditions (Fig. 8d,e and Extended Data Fig. 10a). Control studies using mice, without counter-stimulus, asayed with the same series of noxious heat stimuli did not show differences in responses between series of tests (Extended Data Fig. 10c). We next examined whether cVLM TH neurons are required for counter-stimulus-induced analgesia by developing a mouse model. In this model, reactivity to heat was assessed in an injured paw (mild burn) and then a counter-stimulus was delivered (capsaicin injection into a forepaw), comparing the withdrawal responses before and after counter-stimulus (Fig. 8f). Using this model, we found that a counter-stimulus can produce analgesia in the injured hind paw (Fig. 8g). Next, we tested whether chemogenetic inhibition of cVLM TH neurons attenuate counter-stimulus-induced analgesia. In these experiments, we used mice in which we performed chemogenetic inhibition of cVLM TH neurons (Fig. 8h). Demonstrating that the cVLM is required for counter-stimulus-induced analgesia, when the cVLM TH neurons were inhibited, counter-stimulus-induced analgesia was eliminated (Fig. 8I). Together, our results establish that the cVLM circuit is required for counter-stimulus-induced analgesia and is sufficient to induce analgesia. We recently investigated the contribution of output from cVLM TH neurons to the PVT in food-seeking behaviors 34 . These studies demonstrated that the cVLM is activated during extreme glucose privation (glucoprivation). In addition to being involved in homeostatic feeding responses to glucoprivation, it has been reported that glucoprivation itself induces analgesia 5 . Therefore, we wondered whether the cVLM might also be involved in this type of analgesic response. To test this proposal, we induced glucoprivation in mice by administering 2-deoxy-d-glucose (2-DG) and examined whether the resulting antinociceptive responses are attenuated by chemogenetic inhibition of cVLM TH neurons. Indeed, inhibition of VLM TH neurons prevented 2-DG-induced antinociception (Fig. 8j,k) consistent with the participation of the cVLM-LC-SC circuit in glucoprivation-dependent analgesia. However, this does not rule out non-LC-SC circuits being involved.

Discussion
Here we investigated a brain region previously shown to regulate pain, and uncovered molecular details and mechanisms for a cVLM-LC-SC pathway 2,9 . We identified a small group of noradrenergic neurons in the cVLM that are robustly stimulated by noxious stimuli. When activated, these neurons trigger marked antinociception to heat without affecting behavioral responses to all other sensory modalities tested. Animals in which cVLM TH neurons were inhibited or ablated exhibited increased sensitivity to heat, suggesting that these neurons provide tonic inhibition in naïve conditions. Our work further revealed that cVLM TH neurons send projections to the LC, that activation of cVLM TH neurons induces c-Fos labeling of LC TH neurons and that activation   20 . Subsequent studies reported that glutamate and GABA agonists injected into a similar region produced the same effects 21,22 . Although the exact neurons affected by these manipulations remain unclear, our results provide a possible explanation for these findings. As cVLM TH neurons are predominantly excitatory, glutamate injection and electrostimulation might directly activate them to trigger antinociception through the pathways we describe. It is not immediately apparent how the neurons we characterized might be affected by GABA-agonist injection, but possible explanations could be that there are cVLM TH neuron-independent pathways or that inhibitory neurons modulate cVLM TH neuronal activity through disinhibition. Previous studies found connections from the cVLM to SC and from the cVLM to A5 nucleus; however, we did not find evidence that cVLM TH neurons target these areas 13,24 , suggesting there may be additional cVLM neurons involved in pain regulation. We attempted to map the afferent inflow to cVLM TH neurons, because noxious signals are initially detected by sensory for L and P = 0.018 for R hind paws, respectively, two-sided paired t-test; data represent means ± s.e.m. c, Approach used to measure the contribution of LC norepinephrine on cVLM-induced antinociception; AAV-mediated CRISPR was used to disrupt TH expression in the LC. d, Representative image of a coronal pons section showing staining for TH (magenta and DAPI-cyan) in mice treated with indicated viruses; similar results were observed in n = 3 mice. Scale bars, 500 µm. Boxed areas indicate magnified images. Quantification of neurons revealed 83% ± 2.6% of TH-stained neurons were lost following CRISPR treatment (1,362 TH + neurons in control versus 224 neurons). e, There was significant change (left side only) in chemogenic-induced nociception responses in Hargreaves tests of TH-cre mice injected with AAV9-CMV-FLEX-SaCas9-U6-sgRNA-TH bilaterally into the LC, n = 8 mice, P = 0.047 and P = 0.54 for L and R hind paws, respectively, two-sided paired t-test; data are the mean ± s.e.m. GFP control animals (injection of AAV-FLEX-GFP into LC) showed significant changes in responses, n = 8, P = 0.0002 and P = 0.0015, for L and R hind paws, respectively, two-sided paired t-test; data represent means ± s.e.m. f, On the hot plate test (52 °C), the latency to first lick was significantly changed in control mice (LC-GFP) upon chemogenetic stimulation (CNO) of cVLM TH neurons, n = 8 mice, P = 0.024; data are the mean ± s.e.m. Mice injected with AAV9-CMV-FLEX-SaCas9-U6-sgRNA-TH bilaterally into the LC exhibited no change in latency upon chemogenetic activation of cVLM TH neurons, n = 8 mice, P = 0.19, two-sided paired t-test; data are the mean ± s.e.m. g, Experimental approach to assay modulation of behavior elicited by chemogenetic inhibition (DREADDi) of cVLM TH neurons. h, Intrathecal injection of the alpha-2 adrenergic agonist clonidine reversed the CNO-induced sensitization of hind paw responses measured with the Hargreaves tests, n = 7, P = 0.0018 and P = 0.0067, for L and R hind paws, respectively, two-sided paired t-test; data are the mean ± s.e.m. Article https://doi.org/10.1038/s41593-023-01268-w neurons and signals from these neurons are transmitted through SC circuits to the brain. Although we observed cells adjacent to cVLM TH neurons that are postsynaptic to SC projection neurons, we do not know whether these cells activate cVLM TH neurons. Future studies will be required to determine whether these or other inputs transmit signals from noxious insults to cVLM TH neurons. In addition, as well as pain inputs affecting cVLM TH neurons, we showed that glucoprivation evoked antinociception and requires cVLM TH neurons. This indicates that cVLM TH neurons receive input from non-SC sources in addition to indirect input from SC projection neurons, raising the interesting possibility of a function for the cVLM as a center for convergence of different stressful stimuli that effect nociception.
We previously described that the cVLM TH neurons send projections to the PVT and demonstrated that this connection can induce food-seeking behavior 34 . In this study, we show that the cVLM TH terminals in the PVT do not produce antinociception, and that LC terminals in the PVT do not induce food seeking. The most parsimonious explanation of these results is that different cVLM TH projections elicit specific   (gray). e, Quantification of AUC for responses to heat ramps were significantly changed after capsaicin treatment, n = 8 mice, P = 0.033 two-sided paired t-test. f, Experimental design for measurement of counter-stimulus-induced analgesia; one hind paw received a mild burn (ipsilateral) and withdrawal latencies were determined using the Hargreaves test before and after capsaicin injection into a forepaw. g, Latencies for withdrawal of the ipsilateral paw were significantly changed compared to baseline after the mild burn and were significantly increased compared to mild burn after administration of counter-stimulus, n = 11 mice, P < 0.001 and P = 0.035, respectively, two-sided paired t-test; data are means ± s.e.m. h, Approach used to determine the effect of the cVLM TH circuit on counter-stimulus-induced analgesia. i, Chemogenetic inhibition of cVLM TH neurons prevented attenuation of counter-stimulation (capsaicin)-induced analgesia of the ipsilateral paw, n = 10 mice, P = 0.36, two-sided paired t-test; data are the mean ± s.e.m. j, Hargreaves withdrawal responses were significantly changed after 2-DG treatment compared to baseline, P = 0.0047 and P = 0.023 for L and R hind paws, respectively, and 2-DG responses were significantly reduced compared to chemogenetic inhibition (CNO), P = 0.0013 and P = 0.003 for L and R hind paws, respectively, n = 7 mice, two-sided paired t-test; data are the mean ± s.e.m. k, Proposed model for feed-forward inhibition by the cVLM circuit.
Dashed lines indicate proposed pathways.
Article https://doi.org/10.1038/s41593-023-01268-w responses through dedicated downstream circuits, although this does not exclude other explanations. We observed that a large proportion of cVLM TH neurons are activated by painful stimuli, and we saw a similar proportion of neurons stimulated by glucoprivation 34 . This suggests that there are no specific classes of 'nociceptive' neurons and that cVLM output may not be encoded by labeled lines. In the future, it will be interesting to examine the potential functions of cVLM projections, to discover how they encode signals and to investigate the nature of the different afferent inputs to the cVLM. An unexplained feature of a number of descending pathways is the modality-specific inhibition produced when specific populations of neurons are activated. This phenomenon is apparently at odds with the modality-independent effects of global stimulation of the PAG, RVM or LC. Specifically, it has been reported that enkephalinergic RVM projection neurons modulate only mechanosensitive responses 45,46 . In the PAG, excitatory and inhibitory neurons, depending on whether they are inhibited or excited, affect either only mechanosensory or both mechanosensory and heat responses 47 . Furthermore, manipulation of corticospinal neurons reduces mechanical but not heat sensitivity 10 . Our results show that the cVLM-LC-SC pathway is also modality specific; it only modifies responses to heat. It has been argued that these apparent paradoxical findings may result from the segregation, at many neural levels, of signals for thermal and mechanosensory nociception 48-54 . When artificial means are used to experimentally probe specific neural ensembles, this could result in responses not normally seen when the whole PAG, RVM or LC is stimulated, where likely multiple interconnected descending pathways are engaged. If this is the case, then in physiological settings the output of the cVLM pathway would be coordinated with other pathways to produce analgesia, explaining the apparent inconsistency of a LC-SC output on affecting only heat responses. Indeed, this explanation fits well with studies showing that global pain control involves considerable interaction between descending LC noradrenergic and the RVM serotonergic systems 2,55-57 . Also, recent characterization of the LC has shown that it consists of apparent sub-domains that receive and transmit signals differently 16 . As well as anatomical heterogeneity within the LC, patterns of neural activity differ between sub-domains, and this divergent signaling may also explain the modality specificity of the cVLM-LC-SC pathway. Another interesting feature of the cVLM-LC-SC pathway is its slow deactivation rate. It is possible that this is because this pathway uses norepinephrine, which stimulates a slow deactivating metabotropic receptor signaling cascade 58 . This slow deactivation may occur in the SC as we found no evidence in slice physiology studies to suggest slow deactivation occurs in the LC. Interestingly, diffuse noxious inhibitory control produces slow inactivating SC inhibition, and this occurs through norepinephrine mechanisms 56 .
By uncovering antinociception neurons in the medulla and the pathway they are part of, we redefine and update a key element in the supraspinal pain-control systems. This module, together with other pain-control centers, regulates pain by gating primary sensory signals at the level of the SC. We show that the cVLM-LC-SC pathway can elicit marked analgesia and contributes to counter-stimulus-dependent analgesia. Therefore, targeting the cVLM pathway might provide a means to both investigate and treat pain.

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41593-023-01268-w.

Retrograde tracing
Three wild-type mice received stereotaxic injections of FG (2.0%, Fluoro-Gold; Fluorochrome) in the LC and CTB (0.5% Cholera Toxin B subunit, LIST Biological Laboratories) in the PVT. Brain tissues were collected 7 d after surgery and processed for histology. Antibodies against CTB (1:500 dilution; 703/AB_2314252, LIST Biological Laboratories) and FG (1:50 dilution; Fluorochrome) were applied along with anti-TH to identify cVLM noradrenergic neurons that project to PVT (TH-and CTB-positive neurons) or LC (TH-and FG-positive neurons), and cVLM noradrenergic neurons, which send collaterals to both.

Viral-mediated knockout of tyrosine hydroxylase
A guide (GCCAAGGTTCATTGGACGGCGG) specific to TH was used to generate virus (AAV-CMV-FLEX-SaCas9-U6-sgRNA-TH) following the procedures described previously 38 . Virus was stereotaxically injected to the LC of TH-IRES-Cre mice and behaviors were measured after 3 weeks.

Pseudotyped rabies virus tracing
Helper AAVs (AAV2/9-hSyn-FLEX-TVA-P2A-EGFP-2A-oG, 200 nl per side) were bilaterally injected into the LC of TH-IRES-Cre mice. The fluorescent reporter (EGFP), the avian receptor (TVA) and the rabies envelope glycoprotein (G) were specifically expressed in noradrenergic neurons in the LC with a Cre-dependent manner. Pseudotyped rabies virus (EnvA-SAD-∆G-mCherry, 200 nl per side) were bilaterally injected into the same location in the LC 2 months later. The G-deficit pseudotyped rabies virus can only infect the noradrenergic neurons that expressed TVA receptor and glycoprotein G. The infectious viral particles generated in these noradrenergic neurons can trans-synaptically spread to presynaptic neurons that made a monosynaptic projection to LC noradrenergic neurons. For rabies virus tracing in the cVLM, helper AAVs (AA8-CAG-FLEX-TCB and AAV-EF1a-DIO-HB) were unilaterally injected into the cVLM, and pseudotyped rabies virus (EnvA-SAD-∆G-GFP, 200 nl) was injected 4 weeks later.

Stereotaxic surgery
All stereotaxic surgeries were conducted as described in our animal study protocol. Mice were anesthetized with a ketamine/xylazine solution (100 mg/10 mg in PBS) and a stereotaxic device (Stoelting) was used for viral injections at the following stereotaxic coordinates: cVLM, −2.50 mm from lambda, ±1.40 mm lateral from midline, and −5.30 mm vertical from cortical surface. LC, −5.50 mm from bregma, 0.95 mm lateral from midline, and −3.50 mm vertical from cortical surface. PVT, −1.6 mm from bregma, −0.06 mm lateral from midline with a six-degree angle, and −3.0 mm from cortical surface. AAVs were injected with an oil hydraulic micromanipulator (Narishige). AAVs were injected at a total volume of 0.1 µl in the cVLM. All other AAVs were injected at approximately 0.2-0.3 µl. Following stereotaxic injections, AAVs were allowed 2-3 weeks for maximal expression. Optical fibers with diameters of 200 µm (0.48 NA) and 400 µm (0.66 NA) were used for optogenetics and fiber photometry experiments, respectively (Doric Lenses). These fibers were implanted over the cVLM or LC immediately after viral injection and cemented using C&B Metabond Quick Adhesive Cement System (Parkell). Mice received subcutaneous injections with ketoprofen (5 mg per kg body weight) for analgesia and Article https://doi.org/10.1038/s41593-023-01268-w anti-inflammatory purposes pre-operatively and post-operatively and were allowed to recover on a heating pad.

Histology
Mice were euthanized with CO 2 and subsequently subjected to transcardiac perfusion with PBS and then with paraformaldehyde (4% in PBS). Brains were then postfixed in 4% paraformaldehyde at 4 °C overnight, and cryoprotected using a 30% PBS-buffered sucrose solution for ~24-36 h. Coronal brain sections (40 µm) were acquired using a cryostat (CM1860, Leica). For immunostainings, brain sections were blocked in 10% NGS in PBST for 1 h at room temperature (RT), followed by incubation with primary antibodies in 10% NGS-PBST for 24-48 h at 4 °C. Sections were then washed with PBST (3 × 15 min) and incubated with fluorescent secondary antibodies at RT for 1 h in 10% PBST. Sections were washed in PBS (3 × 15 min), mounted onto glass slides and cover-slipped with Fluoromount-G (Southern Biotech, 0100-01). Images were taken using a Nikon C2 + confocal microscope. Image analysis and cell counting were performed using ImageJ software by a blinded experimenter (Fiji, version 2017 May 30).

Fos expression
For c-Fos expression upon capsaicin or ATP administration to plantar skin, mice were anaesthetized with isoflurane (2%) for 5 min. PBS containing 10 nmol of capsaicin or 500 nmol of ATP were injected into the left hind paw of wild-type mice and then mice were returned to the home cage. Brain tissues were collected 1 h after injection and subjected to c-Fos immunohistochemistry analysis. For c-Fos expression in mice with AAV2-DIO-DREADDq-mCherry virus injection, brain tissues were collected 1 h after intraperitoneal injection of CNO.

Bulk Ca 2+ and fiber photometry
Fiber photometry procedures and calcium measurements were performed by following methods previously described 34 . Mice were first allowed to adapt to the experimental chambers and the attached fiber patch cord for 60 min before each testing session. A fiber photometry system (Doric Lenses) was used to record fluorescence signals. The system is integrated with two continuous sinusoidally modulated LEDs (DC4100, ThorLabs) at 473 nm (211 Hz) and 405 nm (531 Hz), which served as light source to excite GCaMP6s and an isosbestic autofluorescence signal, respectively. Fluorescence signals were collected by the same fiber implant that was coupled to a 400-µm optical patch cord (0.48 NA) and focused onto two separate photoreceivers (2151, Newport Corporation). The RZ5P acquisition system (Tucker-Davis Technologies), equipped with a real-time signal processor controlled the LEDs and also independently demodulated the fluorescence brightness from 473 nm and 405 nm excitation. The LED intensity (range 10-15 µW) at the interface between the fiber tip and the animal was constant throughout the session. All photometry experiments were performed in behavioral chambers, square enclosures on the hot plate (IITC Life Science) or mouse enclosures for Plantar Test Instrument (Ugo Basile). For ΔF/F analysis, a least-squares linear fit to the 405-nm signal to align it to the 470-nm signal was first applied. The resulting fitted 405-nm signal was then used to normalize the 473-nm signal as follows: ΔF/F = (473-nm signal − fitted 405-nm signal) / fitted 405-nm signal. For counter-stimulus experiments, mice were tested three times with 25-55 °C ramps, mice were given a 1-h rest and then injected with capsaicin (counter-stimulus). Next, a further three heat ramp trials were performed.

Combined optogenetic stimulation of LC terminals and photometry of LC TH neurons
We used one optical fiber as described for photometry experiments. This fiber was connected to a six-port fluorescence mini cube (Dori-cLens), which allowed combined isosbestic excitation (400-410 nm), GCaMP excitation (460-490 nm) and emission (500-550 nm), and red fluorophore excitation (540-570 nm). Three light sources were used, a 405-nm LED for isosbestic excitation, a 473-nm LED for GCaMP6s excitation and a 561-nm laser for Chrimson excitation. Two separate photoreceivers collected isosbestic and GCaMP6s signals (2151, Newport Corporation). The intensity of illumination from the 473-nm LED was constant throughout the session and was adjusted to a minimal level to detect GCaMP6s signal (10-15 µW at the interface between the fiber tip). Activation of Chrimson was previously reported to occur from the excitation of GCaMP 59 , and this effect would increase the baseline and reduce the signal-to-noise ratio of GCaMP6s signals. Therefore, illumination for GCaMP6s was reduced to a minimum to decrease this effect.

Optogenetics
TH-IRES-Cre mice injected with either Cre-dependent ChR2 or Cre-dependent GFP (control) in the cVLM and an optical fiber placed above cVLM were behaviorally tested 3 weeks later. Mice were tethered with an optical patch cord and placed in the Perspex enclosure (10 cm × 10 cm × 15 cm) with free movements. After habituation for 60 min, Hargreaves tests were performed to measure the baseline of hind paw withdrawal latency. Then, mice received light stimulation with a blue LED (470 nm; Thorlabs, M470F1) at a frequency of 20 Hz (10 ms width) for 2 min. Hargreaves tests were carried out to measure the hind paw withdrawal latency during the stimulation and at 2 min, 5 min, 10 min, 20 min and 30 min after cessation of stimulation. For optical activation with Chrimson, a 561-nm laser (Opto Engine, 561-50 mW) was used to generate light stimulation at a frequency of 20 Hz (10 ms width) for 2 min during which Hargreaves tests were performed.

Mouse behavioral measurements
All behavioral experiments were conducted during the light cycle at ambient temperature (~23 ˚C). For all behavioral paradigms, the experimenter was blinded to the genotype of mice under study. Ear-tag numbers were read after experiments and results were unblinded after testing sessions.

Hargreaves test
Mice were habituated to the testing enclosures (Ugo Basile) for 60 min. Habituation was repeated for 2 d. On testing day, after the mice were acclimatized for 60 min in the testing enclosure, a radiant heat beam was applied to the center of the hind paw and reaction time between the start of the heat stimuli and lifting the hind paw was recorded as the hind paw withdrawal latency. A cutoff time of 15 s was used to prevent tissue damage. Consecutive tests of the same paw were separated by at least 3 min. The test was repeated for five trials for both left and right hind paws. The averages of the withdrawal latencies were calculated. Mild burn was achieved by placing the hind paw, while mice were deeply anesthetized, in a water bath at 55 °C for 15 s.

Cold plantar test
Cold responses were tested as described previously 60 . Briefly, a dry ice pellet was applied below the hind paw of a mouse sitting on a glass surface and time to withdrawal was measured. Withdrawal was tested five times for each hind paw, and consecutive tests of the same paw were separated by at least 3 min.

Itch test
Behavioral assessment of scratching behavior was conducted as described previously 50 . Briefly, mice were injected subcutaneously Nature Neuroscience Article https://doi.org/10.1038/s41593-023-01268-w into the nape of the neck with chloroquine. Compounds were diluted in PBS. Scratching behavior was recorded for 30 min and is presented in numbers of bouts observed in 30 min. One bout was defined as scratching behavior toward the injection site between lifting the hind leg from the ground and either putting it back on the ground or guarding the paw with the mouth. Injection volume was always 10 µl.

Von Frey test
Mechanical sensitivity thresholds were assessed using calibrated von Frey filaments using the simplified up-down method. Animals were acclimatized in a plastic cage with a wire mesh floor for 1 h and then tested with von Frey filaments with logarithmically incremental stiffness (starting with 0.4 g). Each filament was applied for 5 s, and the presence or absence of a withdrawal response was noted. The filament with the next incremental stiffness was then applied, depending on the response to the previous filament, and this was continued until there were six positive responses. The filaments were applied to the glabrous skin on the hind paw, and a positive response was recorded when there was lifting or flinching of the paw. The force required for 50% withdrawal was determined by the up-down method.

Rotarod test
Motor coordination was tested by measuring the performance on an accelerating rotarod (IITC Life Science) with the rod programmed to accelerate from 4 to 40 r.p.m. over 5 min. During the experimental testing session, the mice were allowed two trial runs followed by four test runs and the average of the maximum r.p.m. tolerated was recorded.
For each mouse, the maximum times on the rota-rod were averaged.

Hot plate test
Hot plate tests (IITC Life Science) were used to assess the nociception upon a high-temperature stimulus. The latency to lick a hind paw when the mouse was placed on a 52.0 °C hot plate was measured. The plate was enclosed with four Plexiglas walls and a lid so that the mouse could not escape. The mouse was removed from the plate after 30 s.

Randall-Selitto test
A modified Randall-Selitto device (IITC Life Science) was used to automatically measure the responses when pressure was applied to the tail. The mouse was placed into a mouse restrainer with the tail exposed to access with a handheld probe. Pressure was applied to the tail until a response was observed. The maximum force applied during the test was recorded.

Feeding behavior
As described in a previous paper 34 , TH-IRES-Cre mice injected with either Cre-dependent ChR2 or Cre-dependent mCherry (control) in the cVLM and an optical fiber placed above the pPVT or LC were behaviorally tested 3 weeks later. First, mice were tethered with an optical patch cord and placed in an open-field box (45 × 45 × 40 cm) where they were given access to 20 mg food pellets for 30 min (pre-test). Immediately after the pre-test, mice received light stimulation with a blue laser tuned at 473 nm at a frequency of 20 Hz (duration, 10 ms) for 30 min using a 1 min 'ON'/2 min 'OFF' protocol (stimulation). After light stimulation, mice were given another 30 min with access to food (post-test). In addition, for these mice, the duration, quantity and timing of feeding epochs were quantified using a custom-designed feeding experimentation device (FED3). The power of the blue laser for all experiments was 5-10 mW, measured at tip of the patch cord. Chrimson. An LED (Lumen 300-LED, Prior Scientific) was used to activate jGCaMP7s. Images obtained before light stimulation served as baseline, and the fluorescence changes of jGCaMP7s after light stimulation were analyzed with ImageJ. TTX (1 µM) and 4-AP (100 µM) were applied to isolate the monosynaptic response. To isolate the noradrenergic or glutamatergic effects on GCaMP responses, initial recordings were made in ACSF to establish a baseline response, then selective antagonists were applied via the bath. For slices from three different mice, we applied via the bath the beta-adrenergic receptor antagonist propranolol (10 µM) and recorded GCaMP responses at least 10 min after initial application of the antagonist. For slices from a separate set of three mice, we simultaneously applied via the bath the AMPA receptor agonist NBQX (10 µM) and the NMDA receptor antagonist AP5 (50 µM) and recorded GCaMP responses after at least 10 min had passed since initial application of the antagonists. All antagonists were purchased from Tocris (Bio-Techne).

Statistics and reproducibility
Prism 8.0 (GraphPad) was used for statistical analyses. Differences between mean values were analyzed using an unpaired two-tailed Student's t-test. Differences were considered significant for *P < 0.05, **P < 0.001, ***P < 0.001 and exact P values are given in the respective figure legend. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications 10,15,34,47 . Data distribution was assumed to be normal but this was not formally tested. Data collection and analyses were not performed blind to the conditions of the experiment and randomization was not used. No data were excluded from data analysis except where post hoc analysis revealed viral transgene expression was absent in the intended site of injection and where animals were removed from study for humane health reasons. Both criteria were preestablished.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.  Fig. 2 A. Injection of AAV22-hSyn-DIO -GFP into TH-CreER mice did not alter CNO evoked behavioral responses in Hargreaves tests, n = 6 mice, p = 0.4031 for left L and p = 0.47, two-sided unpaired t-test, data are presented as mean ± SEM. B. For both male and female mice, withdrawal latencies were significantly increased, in Hargreaves tests, after chemogenetic activation of cVLM TH neurons (CNO administration) compared to saline injected mice (L and R indicate left and right hind-paws respectively), n = 8 male mice, p = 0.0004 for L and p < 0.0001 for R hind-paws; n = 8 female mice, p = 0.0016 for L and t = 7.35, p = 0.0002 for R hind-paws, two-sided paired t-test, data are presented as mean ± SEM. data represent means ± SEM. There were no significant differences in responses between male and female mice, p = 0.63 for L and p = 0.95 for R hindpaws, two-sided unpaired t-testdata are presented as mean ± SEM. C. Hargreaves test responses of mice before and after administration of tamoxifen (to induce translocation of CreERT2 and recombination) were not significantly different, n = 8 mice, p = 0.42 for L and p = 0.089 for R hind-paws, two-sided paired Student T-test, data are presented as mean ± SEM. D. For both male and female mice withdrawal latencies were significantly decreased, in Hargreaves tests, after chemogenetic inhibition of cVLM TH neurons (CNO administration) compared to saline injected mice, n = 3 male, p = 0.041 for L and p = 0.023 for R hind-paws; n = 8 female mice, p = 0.0026 for L and p = 0.0033 for R hind-paws, two-sided paired t-test, data are presented as mean ± SEM. There were no significant differences in responses between male and female mice. p = 0.072 for L and, p = 0.38 for R hind-paws. E. Hargreaves test responses of mice before and after administration of tamoxifen. The were no significant differences between treatment groups, n = 5, p = 0.30 for L and p = 0.078 for R hind-paws, two-sided unpaired t-test, Data are presented as mean ± SEM. Fig. 4 | Effects of chemogenetic activation of cVLM TH -neurons on itch, touch, cold, motor co-ordination and body temperature. Related to Fig. 2. A-F Analysis of behavioral responses in TH-CreER mice injected unilaterally in the cVLM with AAV2-hSyn-DIO-hM3D(Gq)-mCherry and tested in behavioral assays following chemogenetic activation of cVLM TH -neurons (CNO). A. Number of scratching bouts over 30 minutes to intradermal injection of chloroquine (200 µg) in the nape of the neck was not significantly different between treatment groups (±CNO) n = 8 mice, p = 0.76, two-sided paired t-test, data are presented as mean ± SEM. B. Threshold responses to von Frey filament stimulation was not significantly different between treatment groups (±CNO) n = 8 mice, p = 0.095 for L and p = 0.80 for R hind-paws, two-sided paired t-test, data are presented as mean ± SEM. C. Mechanical pinch responses (Randal Selitto method) were not significantly different between treatment groups (±CNO) n = 8 mice, p = 0.47,, two-sided paired t-test, data are presented as mean ± SEM. D. Latencies for withdrawal in plantar reflex responses to cold stimulation were not significantly different between treatment groups (±CNO) n = 8 mice, p = 0.11 for L and p = 0.796 for R hind-paws,, two-sided paired t-test, data are presented as mean ± SEM. E. Motor coordination was not significantly different between treatment groups (±CNO) n = 8 mice, p = 0.43, two-sided paired t-test, data are presented as mean ± SEM. F. Core body temperature measured with a rectal thermal probe was not significantly different between treatment groups (±CNO) n = 5 mice, p = 0.58, two-sided paired t-test, data are presented as mean ± SEM.

Extended Data
Article https://doi.org/10.1038/s41593-023-01268-w Extended Data Fig. 5 | Effects of chemogenetic inhibition of cVLM TH -neurons on itch, touch, cold, motor co-ordination and body temperature. Related to Fig. 2. A-F Analysis of behavioral responses in TH-CreER mice injected unilaterally in the cVLM with AAV2-hSyn-DIO-hM4D(Gi)-mCherry and tested in behavioral assays following chemogenetic inhibition of cVLM TH neurons (CNO). A. Number of scratching bouts over 30 minutes to intradermal injection of chloroquine (200 µg) in the nape of the neck was not significantly different between treatment groups (±CNO) n = 6 mice, p = 0.83, two-sided paired t-test, data are presented as mean ± SEM. B, Threshold responses to von Frey filament stimulation was not significantly different between treatment groups (±CNO) n = 11 mice, p = 0.30 for L and p = 0.55 for R hind-paws, two-sided paired t-test, data are presented as mean ± SEM. C. Mechanical pinch responses (Randal Selitto method) were not significantly different between treatment groups (±CNO) n = 6 mice, p = 0.75, two-sided paired t-test, data are presented as mean ± SEM. D. Latencies for withdrawal in plantar reflex responses to cold stimulation were not significantly different between treatment groups (±CNO) n = 5 mice, p = 0.64 for L and p = 0.08 for R hind-paws,, two-sided paired t-test, data are presented as mean ± SEM. E. Motor coordination was not significantly different between treatment groups (±CNO) n = 5 mice, p = 0.28 for light ON and p = 0.13 for R hindpaws, two-sided paired t-test, data are presented as mean ± SEM. F. Core body temperature measured with a rectal thermal probe was not significantly different between treatment groups (±CNO) n = 6 mice, p = 0.41, two-sided paired t-test, data are presented as mean ± SEM. Representative example of calcium responses of cVLM TH neurons, measured using in vivo fiber photometry, from a single mouse to repeated noxious heat stimulation (on a hot plate; temperature ramps indicated above trace) before and after injection of capsaicin counterstimulus into the fore paw (indicated with red arrow). A 1-hour rest period was included between naïve and counter-stimulus trials. B. Behavioral responses to heat challenge (heat ramp to 55 °C) on a hot plate before and after injection of capsaicin in the forepaw; latency to first lick (left columns). There was a significant difference between trial groups for first lick (±capsaicin), n = 6 mice, p = 0.043, two-sided paired t-test, data are presented as mean ± SEM. C. Averaged in vivo photometry responses of cVLM TH neurons for three trials (averaged) before and after a 1-hour rest period to heat challenges (3 x heat ramp to 55 °C) on a hot plate, showed that averaged responses were not altered to repeated noxious thermal insult or by the 1-hour rest period.

Corresponding author(s): Dr Mark Hoon
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