https://doi.org/10.5061/dryad.kkwh70s82

We identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. With fiber photometry, optogenetics and animal behavior, we demonstrated that these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord upon activation.

Description of the data and file structure

1. Data are the source data for figures and images published in the following paper. 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
2. Source data for bar graphs were organized in the excel files. Data in “*.xlsx” for each figure were named accordingly. For panels in each figure, the data are in the sheets of the excel file named accordingly. For example, the data for Figure 1 panel C(Figure 1C) can be found in sheet “Fig1C” in “Figure1.xlsx” file.
3. In the behavior test data, columns are repeated measures. Each row represents data from one mouse. Five repeated measurements for both left (L) and right (R) hindpaw (for Hargreaves test and cold plantar). L: left hindpaw; R: right hindpaw; Veh: Vehicle; CNO: Clozapine N-oxide (a synthetic ligand for human muscarinic engineered receptors, designer receptors activated by designer drugs or DREADD). Veh L: Left hindpaw measurements after Vehicle injection. Veh R: Right hindpaw measurements after Vehicle injection. CNO L: Left hindpaw measurements after CNO injection. CNO R: Right hindpaw measurements after CNO injection.
4. In feeding experiments, each column represents data from one mouse.
5. In slice GCaMP7s (synthetic fusion of green fluorescent protein (GFP) and calmodulin (CaM), genetically encoded calcium indicator) experiments, data from individual cell were presented. Cells were from three animals.
6. For quantification of images, + and - in the spreadsheets refers to the presence or absence of detectable fluorescent signal from immunostaining.
7. In in vivo fiber photometry experiments, each time point (first column of the data sheet, each time point represents 5 seconds) represents average GCaMP6s signals in five seconds. Each column represents response from one animal.

Figure 1.The cVLM^TH, a brainstem nucleus activated by capsaicin.

Figure1.xlsx includes the data in figure 1c,d,f and h

• Sheet”Fig1C_cfos_CAP+PBS”: Quantification of the percentage of c-Fos⁺TH⁺ to TH+ neurons after capsaicin (CAP) or PBS (Phosphate-buffered saline) injection to the hind paw. Ipsi-capsaicin, Contra-capsaicin: ipsilateral or contralateral cLVM to capsaicin injected hindpaw respectively. Ipsi-PBS, Contra-PBS: ipsilateral or contralateral cLVM to PBS (work as a control) injected hindpaw respectively. TH⁺: Tyrosine hydroxylase immunostaining positive. cLVM: caudal ventral medulla; c-Fos⁺: c-Fos (marker of neuronal activity) immunostaining positive.
• Sheet ”Fig1D_cfos_ATP”: Quantification of the percentage of c-Fos⁺TH⁺ to TH⁺ neurons after ATP or PBS to the hindpaw. Ipsi-ATP, Contra-ATP: ipsilateral or contralateral cLVM to ATP (adenosine triphosphate) injected hindpaw. Ipsi-PBS, Contra-PBS: ipsilateral or contralateral cLVM to PBS (work as a control) injected hindpaw respectively.
• Sheet ”Fig1F_vGlut2_vGat_insitu”: Quantification of the expression of Vglut2 (vesicular glutamate transporter) and Vgat (Slc32a1, vesicular inhibitory amino acid transporter) in TH neurons with RNAscope in situ hybridization. TH⁺vGlut2^+: percentage of vGlut2⁺ vs TH⁺; TH⁺vGAT^+: percentage of vGAT⁺ vs TH⁺.
• Sheet” Fig1H_AAV-cre_Ai9, #of tdT+”: Quantification of labeled anterograde neurons with anterograde transfer of AAV1 to VLM^TH neurons. Data are the numbers of neurons in each mouse. tdTomato⁺: number of tdTomato positive neurons. TH⁺: number of TH⁺ neurons. TH⁺/tdTomato⁺: number of TH and tdTomato positive neurons.

Figure 2: cVLM^TH neurons are activated by noxious stimuli.

Figure2.xlsx: includes the data in figure 2b-f.

• Sheet”Fig2B-Gcamps_CAPinjection”: Averaged in vivo fiber photometry results from cVLM^TH neurons upon stimulation with capsaicin and PBS. CAP: Capsaicin injection into the hindpaw; PBS: PBS injection into the hindpaw. The first column represents the time points (each time points represents 5 seconds) for the measurements. Each column represents data from one animal (numbers are animal ID). Each data point represents averaged fluorescence signal in 5 seconds. CAP or PBS were injected at 15 mins (data point 181 for each mouse). Each data point represents averaged fluorescence signal in 5 seconds.
• Sheet”Fig2C_AUC”: Area under the curve (AUC) quantification showed that capsaicin treatment significantly altered calcium responses compared to PBS-injected controls. Summary of the area under the curve from 15 mins (Capsaicin: capsaicin injection) to 45 mins (30 mins after capsaicin injection, from data point 182 to 542). Capsaicin: data from Capsaicin injection. PBS: data from PBS injection.
• Sheet”Fig2D_Gcamp6s_temp_ramp25-55”: Responses to heat challenge on a hot plate. GCaMP6s: animals with AAV-flex-GCaMP6s virus; GFP: animals with AAV-flex-GFP virus as control for GCaMP6s. Each column represents data from one animal (numbers are animal ID). GCaMP6s-AUC/GFP-AUC: Summary of the area under the curve from 3 mins (temperature ramp start at 25^oC) to 6 mins (temperature ramp stop at 55^oC) from animals with AAV-flex-GCaMP6s or AAV-flex-GFP.
• Sheet”Fig2E_temp_ramp_25-55_heatmap”: Heat map traces from six individual animals to heat challenge and for comparison changes in fluorescence observed in cVLM^TH neurons expressing GFP-expressing control animals. Same data set in Fig2D, but plotted heatmap to show the data from six individual animals. GCaMP6s: animals with AAV-flex-GCaMP6s virus; GFP: animals with AAV-flex-GFP virus as control for GCaMP6s.
• Sheet” Fig2F_Gcamp6s_tailbulldog”: Calcium responses to bulldog clamp on the tail. Similar to Fig2D. Bulldog was applied at 3 mins. GCaMP6s: animals with AAV-flex-GCaMP6s virus; GFP: animals with AAV-flex-GFP virus as control for GCaMP6s. Each column represents data from one animal (numbers are animal ID). GCaMP6s-AUC/GFP-AUC: Summary of the area under the curve from 3 mins to 4 mins from animals with AAV-flex-GCaMP6s or AAV-flex-GFP.

Figure 3: VLM^TH neurons potently control nociceptive behavioral responses.

Figure3.xlsx: includes the data in figure 3d, 3f, 3g-l.

• Sheet”Fig3D_DREADDq expression”: Quantification of the percentage of cVLM^TH neurons expressing DREADDq-mCherry in the cVLM. Each row represents one animal.
• Sheet” Fig3F_cfos_DREADDq_CNO”: Quantification of the percentage of TH neurons expressing c-Fos. Animal were injected with intraperteal injection (i.p.) CNO (CNO) or Vehicle (Veh) solution. Each row represents one animal.
• Sheet” Fig3G_Hargreaves test_DREADDq”: Withdrawal latencies (in seconds) in Hargreaves tests with CNO or Vehicle (L and R indicate left and right hindpaw, respectively in all behavior data). Each row represents one animal (in all behavior data). Five repeated measurements for each hindpaw (for Hargreaves test and cold plantar). M: male mouse; F: female mouse. ADD). Veh L: Left hindpaw measurements after Vehicle injection. Veh R: Right hindpaw measurements after Vehicle injection. CNO L: Left hindpaw measurements after CNO injection. CNO R: Right hindpaw measurements after CNO injection.
• Sheet” Fig3H_leftside vs rightside”: Withdrawal latencies (in seconds) in Hargreaves tests for chemogenetic activation of cVLM^TH neurons (CNO administration) comparing left versus right cVLM injection. Veh L R: Left hindpaw measurements with virus injection in the right side of the cVLM after Vehicle injection. Veh R R: Right hindpaw measurements with virus injection in the right side of the cVLM after Vehicle injection. Veh L L: Left hindpaw measurements with virus injection in the left side of the cVLM after Vehicle injection. Veh R L: Right hindpaw measurements with virus injection in the left side of the cVLM after Vehicle injection. The column titles apply to measurements after CNO injection.
• Sheet” Fig3I_hotpate for DREADDq”: Latency to lick (in seconds) on the hot plate test (52 °C) with CNO or vehicle (Veh) injection.
• Sheet”Fig3k_DREADDi”: Withdrawal latencies (in seconds) in Hargreaves tests with CNO or Vehicle in animals expressing DREADDi. DREADDi: Designer Receptors Exclusively Activated by Designer Drugs coupled to Gi (inhibition upon CNO application). Veh L/R: Left/right hindpaw measurements after Vehicle injection. CNO L/R: Left/right hindpaw measurements after CNO injection.
• Sheet”Fig3l_DTA”: Withdrawal latencies (in seconds) in Hargreaves tests with CNO or Vehicle in animals expressing DTA (diphtheria toxin subunit A ). Pre-Tam L/R: Left/right hindpaw measurements before tamoxifen treatments (without DTA expression, work as baseline control); Post-Tam L/R; Left/right hindpaw measurements two weeks after tamoxifen treatments (after DTA expression).

Figure 4: Optogenetic stimulation of cVLM^TH triggers extended suppression of nociceptive responses.

Figure4.xlxs: includes the data in figure 4b-d

• Sheet “Fig4b_control_optic”: Withdrawal latencies (in seconds) in Hargreaves tests with optogenetic stimulation. AAV2-DIO-GFP virus was injected into cVLM of TH-Cre mice as control. Light OFF L/R: left/right hindpaw without optogenetic stimulation (as control); Light ON L/R; left/right hindpaw with 20Hz optogenetic stimulation.
• Sheet “Fig4c_Chr2_optic”: Withdrawal latencies (in seconds) in Hargreaves tests in animals with AAV2-DIO-ChR2-EYFP injection. Light OFF L/R: left/right hindpaw without optogenetic stimulation (as control); Light ON L/R; left/right hindpaw with 20Hz optogenetic stimulation.
• Sheet “Fig4d_ChR2_time course”: Withdrawal latencies (in seconds) in Hargreaves tests. Baseline: measurements without optical stimulation (as control); 0 min (during stimulation); measurements during 20Hz optical stimulation; 2/5/10/20/30 min; 2/5/10/20/30 mins after 20Hz optogenetic stimulation.

Figure 5:A descending antinociceptive cVLM–LC–SC circuit.

Figure5.xlxs: includes the data in figure 5f, 5j-k, 5m-n

• Sheet”Fig5f_cfos in LC”: Quantification of numbers of LC (locus ceruleus) neurons projecting to the SC (GFP⁺TH⁺) that were c-Fos positive (c-Fos⁺GFP⁺TH⁺) with CNO or Vehicle (Veh) in mice with DREADDq expressed in cLVM.
• Sheet”Fig5J_cLVM_Chrimson_LC_GCaMP6s”: Measurements of calcium responses in LC neurons using in vivo fiber photometry. cLVM-Chrimson: data from mice with cVLM^TH neurons expressing ChrimsonR-tdTomato. mCherry: data from control animals with cVLM^TH neurons expressing mCherry. Each column represents average data from one animal.
• Sheet”Fig5M_ChrimsonChR2 LVM_stimLC”: Withdrawal latencies (in seconds) in Hargreaves tests with optogenetic stimulation of Chrimson in LC terminals. Light OFF L/R: measurements of left/right hindpaw without optical stimulation (as control); Light ON L/R: measurements of left/right hindpaw with 20Hz optogenetic stimulation. Each row represents five repeated measurements from one animal.
• Sheet”Fig5N_mCherryLVM_stimLC”: Control experiments similar to Figure5M. Optogenetic stimulation of LC terminals expressing mCherry. Light OFF L/R: measurements of left/right hindpaw without optical stimulation (as control); Light ON L/R: measurements of left/right hindpaw with 20Hz optogenetic stimulation. Each row represents five repeated measurements from one animal.

Figure 6: cVLM^TH neurons form monosynaptic inputs in the LC.

Figure6.xlxs: includes the data in figure 6c, 6e

• Sheet”Fig6c_LC slice GCaMP6s”: average intensity peak of jGCaMP7s fluorescence relative to baseline fluorescence from cells in the ventral LC. Individual neurons data from three mice were included. ACSF: Baseline; TTX: TTX was applied in ACSF; TTX+4AP: TTX and 4AP were applied in ACSF.
• Sheet” Fig6e_LC slice dorsal ventral” : Quantification of the averaged intensity peaks of GCaMP7s fluorescence relative to baseline fluorescence for cells located in the ventral or dorsal LC. vLC: measurements for cells from ventral LC; dLC: measurements for cells from dorsal LC.

Figure 7: Antinociceptive and pro-nociceptive responses induced by cVLM^TH neurons are mediated via noradrenergic mediated processes.

Figure7.xlxs: includes the data in figure 7b, 7e, 7f, 7h,

• Sheet”Fig7b_Yohimbine“: Withdrawal latencies in Hargreaves tests in mice expressing DREADDq in cLVM with intrathecal (i.t.) administration of yohimbine. Yohimbine L/R: Left/right hindpaw measurements with i.t. injection of Yohimbine. CNO + Yohimbine L/R: Left/right hindpaw measurements with i.p. injection of CNO and i.t. injection of Yohimbine.
• Sheet"Fig7E_cLVMDREADDq_LCTHgRNA": Withdrawal latencies in Hargreaves test in mice expressing DREADDq in cLVM and expressing TH guild RNA(gRNA, AAV9-CMV-FLEX-SaCas9-U6-sgRNA-TH) or control (GFP) in LC. Each row represents one animal with GFP(mouseID-GFP) and or animal with gRNA (mouseID-gRNA).
• Sheet"Fig7f_THgRNA_hoteplate": The latency to lick (in seconds) on the hot plate test (52 °C) with CNO or PBS (vehicle) in mice expressing TH guild RNA(gRNA) or control (GFP). Each row represent one animal. Veh/CNO-GFP: measurements from mice expressing GFP after vehicle or CNO injection. Veh/CNO-gRNA: measurements from mice expressing gRNA after vehicle or CNO injection.
• Sheet"Fig7h_DREADDi_cloidine": Withdrawal latencies in Hargreaves test in mice expressing DREADDi in cLVM with intrathecal (i.t.) administration of clonidine. Clonidine L/R: Left/right hindpaw measurements with i.t. injection of Clonidine. CNO + Clonidine L/R: Left/right hindpaw measurements with i.p. injection of CNO and i.t. injection of Clonidine.

Figure 8: The cVLM circuit is required and sufficient for counter-stimulus-induced analgesia.

Figure8.xlxs: includes the data in figure 8a, 8d, 8e, 8g and 8i-j.

• Sheet”Fig8a_DREADDq_CFA”: Withdrawal latencies in Hargreaves test in mice expressing DREADDq after inflammation. Untreated Basline/CNO: measurements before Freund's adjuvant (CFA) injection into the hind plantar with vehicle or CNO injection; CFA treated: measurements after CFA injection into the hind plantar with vehicle injection; CFA treated + CNO: measurements after CFA injection into the hind plantar with CNO injection.
• Sheet”Fig8d_cLVMGCaMP6s_forepaw_CAP”: Averaged responses over trials before and after counter-stimulation after subtraction of baselines in fiber photometry experiments. Each column represents data from one animal at different time point. Each data points represent averaged response in five seconds. Heat ramp starts at 3 min and end at 6 min. Capsaicin was injection after first heat ramp and recording resume 1 hour after Capsaicin injection.
• Sheet”Fig8e_AUC”: Quantification of AUC for responses to heat ramps. Before Capsaicin: area under the curve from 3 min to 6 min before Capsaicin injection; After Capsaicin: area under the curve from 3 min to 6 min of the second heat ramp 1 hr after Capsaicin injection.
• Sheet”Fig8g_WT_DNIC”: 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 in wild type mice. baseline ipsi: measurements from ipsilateral hind paw before mild burn; 24 hrs after Mild Burn ipsi: measurements from ipsilateral hind paw 24 hours after mild burn; 1 hr after forepaw Capsaicin ipsi: measurements from ipsilateral hind paw 1 hour after Capsaicin injection into ipsilateral forepaw with 24-hr mild burn.
• Sheet”Fig8I_DNIC_THcreDREADDi”: 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 in mice expressing DREADDi in cVLM^TH neurons. baseline ipsi: measurements from ipsilateral hind paw before mild burn; 24 hrs after Mild Burn ipsi: measurements from ipsilateral hind paw 24 hours after mild burn; 1 hr after forepaw Capsaicin ipsi: measurements from ipsilateral hind paw 1 hour after Capsaicin and CNO injection into ipsilateral forepaw with 24-hr mild burn.
• Sheet”Fig8j_DREADDi_2-DG”: Hargreaves withdrawal responses after 2-DG treatment compared to baseline. 2-DG L/R: measurements from left/right hindpaw with i.p. injection of 2-DG; CNO + 2-DG L/R; measurements from left/right hindpaw with i.p. injection of 2-DG and CNO.

Extended Data Fig. 1 cVLM^TH-neurons are catecholaminergic

sFig1.xlxs: includes the data in Extended Data Fig. 1b, 1d, 1e.

• Sheet”sFig1b_cfos_TRPV1-KO”: Quantification of cfos expression in TH-neurons. Ipsi-WT: ipsilateral side of Wild-type mouse hindbrain with Capsaicin in hindpaw; Contra-WT; contralateral side of Wild-type mouse hindbrain with Capsaicin in hindpaw; Ipsi-TRPV1KO: ipsilateral side of TRPV1 knockout mouse hindbrain with Capsaicin in hindpaw; Contra-TRPV1KO; contralateral side of TRPV1 knockout mouse hindbrain with Capsaicin in hindpaw.
• Sheet”sFig1DE.Vmat,PNMT”: Multilabel ISH of cVLM^TH neurons revealed that the majority of these neurons express monoamine transporter Vmat (Slc18a2) but not adrenaline synthesizing enzyme PNMT. TH⁺vMAT⁺: percentage of vMAT⁺ neurons that are TH⁺; TH⁺PNMT⁺: percentage of PNMT⁺ neurons that are TH⁺. TH⁺DDC⁺: percentage of DDC⁺ neurons that are TH⁺. TH⁺DBH⁺: percentage of DBH⁺ neurons that are TH⁺. tot_th: total number of TH⁺ neurons; th_vmat: number of vMAT⁺ neurons that are TH⁺. th_pnmt: number of pnmt⁺ neurons that are TH⁺. th_ddc: number of DDC⁺ neurons that are TH⁺. th_DBH: number of dbh⁺ neurons that are TH⁺.

Extended Data Fig. 2 Photometry of cVLM^TH-neurons to mild somatosensory stimuli.

sFigure2.xlxs: includes the data in Extended Data Fig. 2b-e

• Sheet “sFig2b_Gcamp6s_PAW brush”: Averaged intracellular calcium responses, using in vivo fiber photometry, of cVLM^TH neurons. Responses to mild mechanical with brush to hind-paw. Each column represent one animal (first 5 columns). Each time point (each cell in the column) is the averaged response in 5 seconds. Baseline(12): area under the curve from 5s-baseline; Stimuli(13): area under the curve from 5s after start of brush.
• Sheet “sFig2c_cLVM_Gcamps6s_Vonfrey1.4”: Averaged intracellular calcium responses, using in vivo fiber photometry, of cVLM^TH neurons. Responses to von Frey filament on plantar surface of hind-paw. Each column represent one animal (first 6 columns). Each time point are averaged response in 5 seconds. Baseline(11): area under the curve from 5s-baseline; Stimuli(12):area under the curve from 5s after start of von frey filament stimulation.
• Sheet “sFig2d_cLVM_Gcamps6s_hargreaves”: Averaged intracellular calcium responses, using in vivo fiber photometry, of cVLM^TH neurons. Responses to localized heating (Hargreaves test on plantar surface of hind-paw). Each column represent one animal (first 6 columns).. Each time point are averaged response in 5 seconds. Baseline(13-14): area under the curve from 10s-baseline; Stimuli(15-16): area under the curve from 10s after start of localized heating.
• Sheet “sFig2e_cLVM_GCaMP6s_cold 30.5-0”: Averaged intracellular calcium responses, using in vivo fiber photometry, of cVLM^TH neurons. Responses to cold plate stimulation. Each column represent one animal (first 6 columns). baseline 3 min(3-39): area under the curve from 3 min-baseline; stimuli 3min (40-75): area under the curve from 3 min during cold plate stimulation.

Extended Data Fig. 3 Control chemogenetic activation and inhibition of cVLM^TH-neurons.

sFigure3.xlxs: includes the data in Extended Data Fig. 3a-e

• Sheet"remove error": blank sheet for removing format error in other sheets.
• Sheet”sFig3a_cLVM_THCreER_CNO GFP con”: Injection of AAV22-hSyn-DIO -GFP into TH-CreER mice did not alter CNO evoked behavioral responses in Hargreaves tests. Veh L: Left hindpaw measurements after Vehicle injection. Veh R: Right hindpaw measurements after Vehicle injection. CNO L: Left hindpaw measurements after CNO injection. CNO R: Right hindpaw measurements after CNO injection.
• Sheet”sFig3b_DREADDq hargreaves MvsF”: For both male and female mice, withdrawal latencies were significantly increased, in Hargreaves tests. Baseline/CNO L/R F/M: Left(L)/right(R) hindpaw measurements after Vehicle(baseline) or CNO injection in female(F) or male(M) mice.
• Sheet”sFig3c_pre-post tamoxifen”: Hargreaves test responses of mice before and after administration of tamoxifen. Pre-tamoxifen baseline L/R: Left/right hindpaw measurements before administration of tamoxifen; Post-tamoxifen baseline L/R: Left/right hindpaw measurements two weeks after administration of tamoxifen.
• Sheet”sFig3d_DREADDi_hargreaves MvsF”: 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. Post-tamoxifen baseline/CNO M/F L/R: Post-tamoxifen: measurements after administration of tamoxifen. baseline/CNO: measurements after vehicle or CNO injection. L/R: Left/right hindpaw, M/F: male/female.
• Sheet”sFig3e_DREADDi pre-post tamoxif”: Hargreaves test responses of mice before and after administration of tamoxifen after chemogenetic inhibition. Pre-tamoxifen/post-tamoxifen baseline L/R: Left/right hindpaw measurements before/after administration of tamoxifen.

Extended Data Fig. 4 Effects of chemogenetic activation of cVLM^TH-neurons on itch, touch, cold, motor co-ordination and body temperature.

sFigure4.xlxs: includes the data in Extended Data Fig. 4a-f

• Sheet”sFig4a_dreaddq-itch”: Number of scratching bouts over 30 minutes to intradermal injection of chloroquine (200 µg) in the nape of the neck. Vehicle: Veh: Number of scratching bouts after Vehicle injection. CNO: Number of scratching bouts after CNO injection.
• Sheet”sFig4b_dreaddq-von frey”: Threshold responses to von Frey filament stimulation was not significantly different between treatment groups (±CNO). Veh L: Left hindpaw measurements after Vehicle injection. Veh R: Right hindpaw measurements after Vehicle injection. CNO L: Left hindpaw measurements after CNO injection. CNO R: Right hindpaw measurements after CNO injection.
• Sheet”sFig4c_dreaddq-randal selittos”: Latencies for withdrawal to mechanical pinch responses (Randal Selitto method). Veh: Number of seconds to mechanical pinch responses after Vehicle injection. CNO: Number of seconds to mechanical pinch responses after CNO injection. Each row represents data from one animal with three repeated measurements.
• Sheet”sFig4d_dreaddq-cold plantar”: Latencies for withdrawal in plantar reflex responses to cold stimulation. Veh L: Left hindpaw measurements after Vehicle injection. Veh R: Right hindpaw measurements after Vehicle injection. CNO L: Left hindpaw measurements after CNO injection. CNO R: Right hindpaw measurements after CNO injection.
• Sheet”sFig4e_dreaddq-rotar rod”: Motor coordination measurements on rotar rod test. Veh/CNO Light On: measurements during light on in the room after Vehicle/CNO injection. Veh/CNO Light OFF: measurements during light off in the room after Vehicle/CNO injection. Each row represents data from one animal with three repeated measurements.
• Sheet”sFig4f_dreaddq-corebody temp”: Core body temperature measured with a rectal thermal probe. Veh: temperature after Vehicle injection. CNO: temperature after CNO injection.

Extended Data Fig. 5 Effects of chemogenetic inhibition of cVLM^TH-neurons on itch, touch, cold, motor co-ordination and body temperature.

sFigure5.xlxs: includes the data in Extended Data Fig. 5a-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).
Data were organized the same way in sFgiure4.
#Sheet”sFig5a_dreaddi-itch”:
#Sheet”sFig5b_dreaddi-von frey”:
#Sheet”sFig5c_dreaddi-radal sellito”:
#Sheet”sFig5d_dreaddi-cold plantar”:
#Sheet”sFig5e_dreaddi-rotar rod”:
#Sheet”sFig5f_dreaddi-core bodytemp”:

Extended Data Fig. 8 cVLM^TH-neurons trigger PVT dependent food-seeking but not analgesia.

sFigure8.xlxs: includes the data in Extended Data Fig. 8a-d, 8f-g

• Sheet”sFig8a&b”: Feeding behavior was measured during optogenetic stimulation of VLM terminals in either the LC or in the PVT. 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. Each column presents data from one animal.
• Sheet”sFig8c_PVT sti_Hargrove test_Ch”: Hargreaves behavioral responses to optogenetic stimulation of VLM terminals in the PVT of TH-Cre mice injected bilaterally with AAV-FLEX-Chrimson. Light OFF L/R: measurements of left/right hindpaw without optical stimulation (as control); Light ON L/R: measurements of left/right hindpaw with 20Hz optogenetic stimulation. Each row represents five repeated measurements from one animal.
• Sheet”sFig8d_collateral”: Quantification of results from three animals with TH, CTB and Fluoro-Gold staining. Number of TH⁺, CTB⁺TH⁺, Fluoro-Gold⁺TH⁺ and Fluoro-Gold⁺, CTB⁺ and TH⁺ neurons were quantified in each animal. TH⁺, CTB⁺ , Fluoro-Gold⁺ represents TH, CTB and Fluoro-gold immuno-positive neurons respectively.
• Sheet”sFig8f_LC Gcamp6s_NBQXAP5”: Glutamate receptors antagonists NBQX and AP5 inhibit cVLM^TH-mediated optogenetic responses in the LC, but β-adrenergic receptor antagonist Propranolol did not. Peak intensity of jGCaMP7s fluorescence for ACSF, and for ACSF with glutamate receptor antagonists (ACSF+NBQX+AP5) was calculated for each neuron. ACSF: measurements in the presence of ACSF only. ACSF+NBQX+AP5: measurements in the presence of ACSF, NBQX and AP5.
• Sheet”sFig8g_LC gcamp6s_propranolol”: peak intensity of jGCaMP7s fluorescence for ACSF, for ACSF with beta blocker Propranolol (ACSF + Propranolol). ACSF: measurements in the presence of ACSF only. ACSF+ Propranolol: measurements in the presence of ACSF and Propranolol.

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

sFigure9.xlxs: includes the data in Extended Data Fig. 9b-d.

• Sheet”sFig9b_Hargrove test_ChR2 LC_cL”: Withdrawal latencies in Hargreaves test with optogenetic activation of cVLM^TH neuron fiber terminals in the LC. Light OFF L/R: measurements of left/right hindpaw without optical stimulation (as control); Light ON L/R: measurements of left/right hindpaw with 20Hz optogenetic stimulation. Light ON + Yohimbine L/R: measurements of left/right hindpaw during optical stimulation with i.t. injection of Yohimbine.
• Sheet”sFig9c_DREADDi_ondansetron”: Withdrawal latencies in Hargreaves test with serotonin 5-hydroytryptamine type 3 (5-HT3) receptor antagonist ondansetron. Veh L/R: Left/right hindpaw measurements after Vehicle injection. CNO L/R: Left/right hindpaw measurements after CNO injection. Ondansetron L/R: Left/right hindpaw measurements with i.t. injection of Ondansetron. CNO + Ondansetron L/R: Left/right hindpaw measurements with i.p. injection of CNO and i.t. injection of Ondansetron.
• Sheet”sFig9d_DREADDi_AS19”: Withdrawal latencies in Hargreaves test with 5-hydroytryptamine type 7 (5-HT7) receptor agonist, AS19. Veh L/R: Left/right hindpaw measurements after Vehicle injection. CNO L/R: Left/right hindpaw measurements after CNO injection. AS19 L/R: Left/right hindpaw measurements with i.t. injection of Ondansetron. CNO + AS19 L/R: Left/right hindpaw measurements with i.p. injection of CNO and i.t. injection of Ondansetron.

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

sFigure10.xlxs: includes the data in Extended Data Fig.10a-c

• Sheet”sFig10a_representative trace”: 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 counter-stimulus into the fore paw (indicated with red arrow). A 1-hour rest period was included between naïve and counter-stimulus trials.
• Sheet”sFig10b_cap lick”: Behavioral responses to heat challenge (heat ramp to 55 °C) on a hot plate before (Lick before CAP) and after (Lick after CAP) injection of capsaicin in the forepaw. Three repeated measurements for each animal. Lick before CAP: latency to lick (in seconds) before CAP injection. Lick after CAP: latency to lick after CAP injection
• Sheet”sfig10c_cLVM_Gcamps6s_new forep”: 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. Each column represents one animal.