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Neurons in the caudal ventrolateral medulla mediate descending pain control

Nature Neuroscience volume 26, pages 594–605 (2023)Cite this article

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Abstract

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.

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Fig. 1: The cVLMTH, a brainstem nucleus activated by capsaicin.
Fig. 2: cVLMTH neurons are activated by noxious stimuli.
Fig. 3: VLMTH neurons potently control nociceptive behavioral responses.
Fig. 4: Optogenetic stimulation of cVLMTH triggers extended suppression of nociceptive responses.
Fig. 5: A descending antinociceptive cVLM–LC–SC circuit.
Fig. 6: cVLMTH neurons form monosynaptic inputs in the LC.
Fig. 7: Antinociceptive and pro-nociceptive responses induced by cVLMTH neurons are mediated via noradrenergic mediated processes.
Fig. 8: The cVLM circuit is required and sufficient for counter-stimulus-induced analgesia.

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Data availability

All data are deposited in an open-source database at https://doi.org/10.5061/dryad.kkwh70s82.

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Acknowledgements

We thank A. Hoover and E. Henry for their help in collecting preliminary data. This work was supported by the intramural research program of the NIDCR, NIH, project ZIADE000721-20 (to M.A.H.) and National Institute of Mental Health, project ZIAMH002950 (to M.P.).

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Authors and Affiliations

  1. Molecular Genetics Section, National Institute of Dental and Craniofacial Research/NIH, Bethesda, MD, USA

    Xinglong Gu, Yizhen Z. Zhang, Caitlynn C. De Preter & Mark A. Hoon

  2. Unit on the Neurobiology of Affective Memory, National Institute of Mental Health, Bethesda, MD, USA

    John J. O’Malley & Mario Penzo

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  1. Xinglong Gu
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Contributions

X.G., M.P. and M.A.H. designed the experiments. X.G. and M.A.H. wrote and edited the paper with input from all authors. X.G., Y.Z.Z., J.J.O. and C.C.D.P. performed experiments and analyzed data. Y.Z.Z. performed and analyzed ISH experiments. C.C.D.P. performed and analyzed c-Fos studies. J.J.O. carried out and analyzed slice physiological experiments. All other studies were performed and analyzed by X.G. M.A.H. and M.P. supervised the project.

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Correspondence to Mark A. Hoon.

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Extended data

Extended Data Fig. 1 cVLMTH-neurons are catecholaminergic.

Related to Fig. 1. A. Representative magnified images, from mice treated with capsaicin, of ventral lateral medulla regions where tyrosine hydroxylase immune-positive neurons (magenta) are found. In control mice, intraplantar application of capsaicin to the hind-paw induced cfos expression (green) prominently in TH-positive neurons. By contrast this treatment, in Trpv1 knockout (Trpv1KO) mice resulted in expression of few cfos-positive neurons. Scale bars: 50 µm. B. Quantification of cfos expression in TH-neurons, n = 6 control mice and n = 3 Trpv1KOmice (Trpv1KO ipsilateral 94/304, Trpv1KO contralateral 92/278; WT ipsilateral 341/452, WT contralateral 219/404), data are presented as mean ± SEM. C. Multilabel ISH of cVLMTH neurons revealed that the majority of these neurons express DDC and DBH. Scale bars: 50 µm. D. Multilabel ISH of cVLMTH neurons revealed that the majority of these neurons express monoamine transporter Vmat (Slc18a2) but not adrenaline synthesizing enzyme PNMT. Scale bars: 50 µm. E. Quantification of the expression of DDC and DBH with TH; from 239 TH+-neurons there were 239 TH-DCC+, 237 TH-DBH+-cells, n = 3 mice. And quantification of the expression of Vmat and PNMT with TH; from 239 TH+-neurons there were 239 TH-Vmat+, 0 TH-PNMT+-cells, n = 3 mice, data are presented as mean ± SEM. F. Using spinal cord injection of AAV1-hSyn-Cre in Ai9 reporter mice together with injection of AAVretro-CAG-GFP, labeled spinal cord anterograde neurons (labeled magenta - tdT) and retrograde labeled spinal cord projecting neurons (labeled green - GFP). Section from caudal medulla (approx. −7.8 Bregma) showed scattered spinal cord anterograde neurons in the cVLM (circled), nucleus of the solitary tract (NTS), and inferior olive complex (IO). Concentrated axonal labeling of corticospinal neurons was seen in the pyramid (py). G. Section from the rostral medulla (approx. −5.7 Bregma) showed anterograde and retrograde labeled neurons in the rostral ventral medulla as well as axonal labeling of corticospinal neurons in the pyramid (py). H. Monosynaptic rabies tracing from cVLMTH-neurons did not reveal pre-synaptic neurons in the spinal cord. I. Adjacent to cVLMTH-starter neurons (red- helper virus and blue TH-immuno-stained) in the medulla were retrograde labeled neurons (green -rabies virus). Note, all red helper virus labeled neurons were TH-positive. However, we note that these neurons could potentially constitute artefacts as non-specific labeled neurons have been reported adjacent to the site of helper virus injections61.

Extended Data Fig. 2 Photometry of cVLMTH-neurons to mild somatosensory stimuli.

Related to Fig. 2. A. Heatmap traces from 3 individual animals to tail clip. B–E. Averaged intracellular calcium responses, using in vivo fiber photometry, of cVLMTH neurons. Responses to mild mechanical with brush to hind-paw (B), von Frey filament on plantar surface of hind-paw (C), localized heating (Hargreaves test on plantar surface of hind-paw) (D), and cold plate stimulation (E) show, only modest increases in intracellular calcium in cVLMTH neurons; n = 6 mice, data are represented as mean results (blue) ± SEM (grey). Quantification of the AUC for measurements are shown to the right of each data set and showed that compared to baseline responses, GCaMP6s responses were not significantly different, p = 0.67, p = 0.997, p = 0. 40, p = 0.31, respectively for panels B-E, two-sided unpaired t-test. Box chart legend: box is defined by 25th, 75th percentiles, whiskers are determined by maximum and minimum, horizontal line represents the mean.

Extended Data Fig. 3 Control chemogenetic activation and inhibition of cVLMTH-neurons.

Related to Fig. 2A. 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 cVLMTH 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 hind-paws, 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 cVLMTH 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.

Extended Data Fig. 4 Effects of chemogenetic activation of cVLMTH-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 cVLMTH-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 Fig. 5 Effects of chemogenetic inhibition of cVLMTH-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 cVLMTH 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 hind-paws, 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.

Extended Data Fig. 6 Innervation of LC by cVLMTH-neurons.

Related to Fig. 3. A–F. Representative images of the pattern of fiber projections from TH-Cre mice injected in the cVLM with AAV9-hSyn-DIO-mCherry-2A-SybGFP, showing synaptic boutons (green) on projecting axons and TH-staining of LC-neurons (blue), n = 3 mice. A–C. Representative image of a sagittal section of the hindbrain showing cVLMTH neuron projections to the LC. D–F. Magnified view of LC. Scale bars, 500 µm in A-C and 50 µm in D–F. 3 V indicates third ventricle.

Extended Data Fig. 7 Innervation of PVT, PVN, BNST, and PAG by cVLMTH-neurons.

Related to Fig. 3. A-G. Representative images of the pattern of fiber projections of TH-Cre mice injected in the cVLM with AAV2-Syn-DIO-TVA-mCherry, showing labeled fibers (magenta), n = 3 mice. A. Representative image of a coronal section of the midbrain showing cVLMTH neuron projections to the PVT and PVN. B. Section through midbrain showing cVLMTH neuron projections to the BNST. C. Section of the hindbrain showing cVLMTH neuron projections to the PAG. D and E. Magnified view of boxed areas in A. F. Magnified view of boxed areas in B. G. Magnified view of boxed areas in C. Scale bars: 1 mm in A-C and 100 µm in D-G. 3 V, LV, D3V, and Aq indicate the third ventricle, lateral ventricle, dorsal third ventricle, and aqueduct respectfully.

Extended Data Fig. 8 cVLMTH-neurons trigger PVT dependent food-seeking but not analgesia.

Related to Figs. 5–6. A-B. Feeding behavior was measured during optogenetic stimulation of VLM terminals in either the LC or in the pPVT. Food intake was quantified in well-fed mice prior to, during and after light stimulation (30 min, pre-test, stimulation and post-test). The stimulation protocol consisted of 30 mins in which light stimulation alternated between 1 min ‘light ON’ (20 Hz) and 2 min ‘light OFF’ bouts. A. Plot of individual performance on feeding behavior during the pre-test, stimulation and post-test for ChR2 (VLM-ChR2) mice. B. Quantification of feeding behavior during VLM-LC and VLM–pPVT stimulation for ChR2 mice. Total food intake in grams, VLM-LC: Pre-Test, 0.048 ± 0.01; Stimulation, 0.035 ± 0.02; Post-Test, 0.06 ± 0.01, n = 8 mice; F (2,23) = 0.623, one-way ANOVA followed by Tukey’s test. Group comparisons: Pre-Test vs Stimulation, p = 0.811; Post-Test vs Stimulation, p = 0.483. for VLM-PVT: Pre-Test, 0.116 ± 0.07; Stimulation, 0.429 ± 0.11; Post-Test, 0.115 ± 0.05, n = 12 mice; F (2,35) = 6.27, one-way ANOVA followed by Tukey’s test. Group comparisons: Pre-Test vs Stimulation, p = 0.02; Post-Test vs Stimulation, p = 0.04. Box chart legend: box is defined by 25th, 75th percentiles, whiskers are determined by maximum and minimum, horizontal lines represent the mean. C. Hargreaves behavioral responses to optogenetic stimulation of VLM terminals in the PVT of TH-Cre mice injected bilaterally with AAV-FLEX-Chrimson. Stimulation did not significantly alter hind-paw withdrawal latency, n = 6 mice, p = 0.42 and p = 0.28 for L and R respectively, two-sided paired t-test, data are presented as mean ± SEM. DE. cVLMTH neurons project collaterally to the PVT and the LC. Retrograde tracers were injected into the PVT (CTB, magenta) and the LC (Fluoro-Gold (FG) green). D. Quantification of results from three animals, data represent means ± SEM. TH+ neurons, n = 440; TH+CTB+ neurons, n = 161; TH+ G+ neurons, n = 107; TH+CTB+FG+ neurons, n = 55. CTB&FG/CTB only + FG only + CTB&FG = 25.6 ± 9.1 mean ± SEM. E. Representative image of the cVLM showing PVT-projecting cVLMTH neurons (TH+CTB+), LC-projecting cVLMTH neurons (TH+FG+) and PVT/LC projecting cVLMTH neurons (TH+CTB+FG+). F. Glutamate receptors antagonists NBQX and AP5 inhibit cVLMTH-mediated optogenetic responses in the LC, but β-adrenergic receptor antagonist Propranolol did not. Z-scores were calculated by using the mean and standard deviation of baseline fluorescence before optical stimulation. Data are presented as the average intensity peak of jGCaMP7s fluorescence relative to baseline fluorescence. Peak intensity of jGCaMP7s fluorescence for ACSF, and for ACSF with glutamate receptor antagonists, p < 0.0001, n = 42 neurons from three animals, two-sided unpaired t-test, data are presented as mean ± SEM. G. Peak intensity of jGCaMP7s fluorescence for ACSF, for ACSF with beta blocker Propranolol, p = 0.23, n = 36 neurons from three animals, two-sided unpaired t-test, data are presented as mean ± SEM.

Extended Data Fig. 9 Controls for optogenetic activation of terminals of cVLMTH-neurons in the LC.

Related to Fig. 7. A. Experimental paradigm used to examine the influence of spinal cord NA on cVLMTH neuron induced antinociceptive effects. B. Optogenetic activation of cVLMTH neuron fiber terminals in the LC induced increased withdrawal latencies in Hargreaves test which were significantly attenuated by intrathecal administration of yohimbine, n = 8 mice, p = 0.0001 for L and p = 0.017 for R hind-paws respectivelytwo-sided paired t-test, data represent means ± SEM. C. Serotonin 5-hydroytryptamine type 3 (5-HT3) receptor antagonist ondansetron (suggested to be responsible, in part, for descending facilitation of pain from the RVM) had no effect on CNO-induced pronociception elicited by chemogenetic inhibition of cVLMTH neuron, n = 7 mice, p = 0.49 for L and p = 0.17 for R hind-paws. D. Similarly, 5-hydroytryptamine type 7 (5-HT7) receptor agonist, AS19 (thought to be responsible, in part, for the descending inhibition of pain from the RVM) also did not affect CNO-induced pronociception, n = 7 mice, p = 0.59 for L and p = 0.0.20 for R hind-paws,two-sided paired t-test, data are presented as mean ± SEM.

Extended Data Fig. 10 Controls for photometry measurements of pain induced analgesia.

Related to Fig. 8. A. Representative example of calcium responses of cVLMTH 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 counter-stimulus 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 cVLMTH 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.

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Gu, X., Zhang, Y.Z., O’Malley, J.J. et al. Neurons in the caudal ventrolateral medulla mediate descending pain control. Nat Neurosci 26, 594–605 (2023). https://doi.org/10.1038/s41593-023-01268-w

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