The anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain1,2,3,4. Projection neurons of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery are channelled through these spinal projection neurons en route to the brain. However, the organizational logic of the anterolateral pathway remains poorly understood. Here we show that two populations of projection neurons that express the structurally related G-protein-coupled receptors (GPCRs) TACR1 and GPR83 form parallel ascending circuit modules that cooperate to convey thermal, tactile and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons. Within this nucleus, axons of spinoparabrachial (SPB) neurons that express Tacr1 or Gpr83 innervate distinct sets of subnuclei, and strong optogenetic stimulation of the axon terminals induces distinct escape behaviours and autonomic responses. Moreover, SPB neurons that express Gpr83 are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Notably, the valence associated with activation of SPB neurons that express Gpr83 can be either positive or negative, depending on stimulus intensity. These findings reveal anatomically, physiologically and functionally distinct subdivisions of the SPB tract that underlie affective aspects of touch and pain.
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The data generated in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
The custom codes used in the study are available at GitHub (https://github.com/SebastianChoi/Choi-et-al-Nature2020) or upon request.
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We thank all members of the Ginty laboratory for discussions and critical feedback during the course of the project; D. Paul and C. Woolf for feedback and critical evaluation of the manuscript; S. Dymecki for providing Rosa26LSL-FSF-TeTx and Rosa26FSF-LSL-SYN-GFP mouse lines; L. Crawford and T. Dickendesher for help with initial characterization of the Calca-FlpE BAC transgenic and AvilFlpO knock-in mouse lines, respectively; C. Cepko for providing AAV1-FLEX-PLAP viruses; S. Arber for providing Cre-dependent AAV expression vector encoding synaptophysin-GFP; C. Guo and the Gene Targeting and Transgenic Facility at the Janelia Research Campus of the Howard Hughes Medical Institute for generating mouse lines; and B. Kun, M. Streeter, C. Breton and O. Gabriel for their assistance with mouse husbandry and histological and behavioural experiments. This work was supported by the Alice and Joseph E. Brooks Funds (S.C.), the Blavatnik Biomedical Accelerator Fund (S.C., D.D.G.), NIH grants NS097344 and AT011447 (D.D.G.), AR063772 (S.E.R.), NS096705 (H.R.K.) and NS111643 (M.G.), the Bertarelli Foundation (D.D.G.), The Hock E. Tan and Lisa Yang Center for Autism Research at Harvard University (D.D.G.) and the Edward R. and Anne G. Lefler Center for Neurodegenerative Disorders (D.D.G). D.D.G. is an investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Nature thanks Robert Gereau, Richard Palmiter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Generation of CreERT2 mouse lines for genetic labelling of anterolateral pathway neurons and Flp mouse lines for labelling of primary sensory neurons.
a–c, Gene targeting strategies used to generate the Robo3IRES-CreERT2 (a), Tacr1CreERT2 (b) and Gpr83CreERT2 (c) mouse lines. a, A 3X-STOP-IRES-CreERT2 cassette was introduced via homologous recombination into the first common coding exon that is shared by different splice variants of the Robo3 gene. b, A CreERT2 cassette was introduced via homologous recombination into the Tacr1 gene, replacing the first coding ATG. c, A CreERT2 cassette was introduced via homologous recombination into the Gpr83 gene, replacing the first coding ATG. IRES, internal ribosome entry site; s.int, synthetic intron; WPRE, Woodchuck hepatitis virus (WHP) posttranscriptional regulatory element; pA, poly(A); f, FRT site; kz, Kozak sequence. d–f, A horizontal section of the lumbar spinal cord. 93.7 ± 2.6% of tdTomato+ neurons were Tacr1+, while 96.6 ± 2.4% of Tacr1+ neurons were tdTomato+. n = 3 mice. g, A transverse section of a Gpr83-GFP mouse. Green and red dots represent GFP and Gpr83 mRNA molecules, respectively, detected with gene-specific RNAscope probes. 96.0 ± 1.2% of GFP+ cells were Gpr83+, while 84.5 ± 5.0% of Gpr83+ cells were GFP+. n = 2 mice. h, Distribution of tdTomato-expressing Robo3+ neurons in the spinal cord dorsal horn (top) and their thalamic projections terminating in the VPL (bottom). n = 2 mice. i–m, Characterization of the AvilFlpO mouse line. n = 4 mice. i–k, The AvilFlpO mouse line labels the majority of DRG neurons (99.0 ± 0.1% of NeuN+ neurons are tdTomato+) (i), nodose ganglia neurons (80.8 ± 5.1% of NeuN+ neurons are tdTomato+) (j) and sympathetic ganglia neurons (98.6 ± 0.3% of TH+ neurons are tdTomato+) (k). l, A transverse section of the vertebral column. tdTomato+ advillin-expressing neurons and their axons are visualized in the spinal cord (asterisk), DRGs (arrows), and sympathetic ganglia (arrowheads). m, A coronal section of the brainstem. tdTomato+ axons of advillin-expressing neurons innervate the nucleus of the solitary tract (arrowhead), the dorsal column nuclei (arrow), and the trigeminal nucleus (asterisk). n–q, Characterization of the Calca-FlpE mouse line. n = 2 mice. A cross section of the lumbar DRG (n–p) and a transverse section of the lumbar spinal cord (q). n–p, 91.9 ± 1.5% of tdTomato+ neurons were CGRP+, while 92.3 ± 1.5% of CGRP+ neurons were tdTomato+. q, tdTomato-expressing axons of CGRP+ DRG neurons are CGRP immunoreactive in the spinal cord dorsal horn.
a, Distribution of EYFP-expressing Tacr1+ (top) or Gpr83+ (bottom) spinal neurons and mCherry-expressing Gad2+ neurons in the superficial lamina of the spinal cord dorsal horn. b, Quantification of % of Gad2-negative neurons in EYFP+ neurons. 97.5 ± 1.4% of Tacr1+ neurons and 99.5 ± 0.5% of Gpr83+ neurons were Gad2-negative. c, Distribution of tdTomato-expressing Tacr1+ neurons and GFP-expressing Gpr83+ neurons in the spinal cord dorsal horn. d, Quantification of co-expression of tdTomato and GFP. 80.2 ± 1.5% and 87.0 ± 2.5% of tdTomato-expressing Tacr1+ neurons are not positive for GFP expression in laminae I and IIo and lamina IIid, respectively. Conversely, 78.0 ± 1.8% and 92.0 ± 1.4% of GFP-expressing Gpr83+ neurons are not positive for tdTomato expression in laminae I and IIo and lamina IIid, respectively. e, Distribution of EYFP-expressing Tacr1+ neurons, Gpr83+ neurons or both in the superficial lamina of the spinal cord dorsal horn. The SPB neurons were retrogradely labelled with CTB injected into the PBNL. Arrowheads, CTB and EYFP double-positive neurons. f, Quantification for % of Tacr1+ SPB neurons, Gpr83+ SPB neurons, and either Tacr1+ or Gpr83+ SPB neurons. g, % of Tacr1+, Gpr83+, Tacr1+ Gpr83+ and Tacr1– Gpr83– SPB neurons calculated from experiments in e, f. h, i, Coronal sections of the ventral brain stem of Tacr1CreERT2 (h) or Gpr83CreERT2 (i) mice whose lumbar spinal cords were injected with AAV1–FLEX–synaptophysin–GFP viruses. MAO, medial accessory olivary nucleus; DAOdf, dorsal accessory olivary nucleus dorsal fold; DAOvf, dorsal accessory olivary nucleus ventral fold; PO, primary olivary nucleus. j, Distribution of tdTomato-expressing Tac1+ neurons in the superficial lamina of the spinal cord dorsal horn. The SPB neurons were retrogradely labelled with CTB injected into the PBNL. Arrowhead, CTB and tdTomato double-positive neuron. k, Quantification of % of Tac1+ SPB neurons. l, Schematic of injections of AAV2-retro-FlpO viruses into the PBNL. m, Distribution of tdTomato-expressing Tacr1+ (left) or Gpr83+ (right) SPB neurons and Tac1+ neurons in the spinal cord dorsal horn. tdTomato (red) and Tac1 (green) mRNA molecules were detected with gene-specific RNAscope probes. Filled arrowheads, double-positive neurons; empty arrowheads, tdTomato+ SPB neurons that do not express Tac1. n, Quantification of co-expression of tdTomato and Tac1 in laminae I and IIo. o, Schematic of lumbar injections of an AAV1–FLEX–synaptophysin–tdTomato virus. p, Distribution of tdTomato-positive synaptic terminals of Tac1+ SPB neurons in the PBNL. q, Quantification of distribution of tdTomato-positive synaptic terminals of Tac1+ SPB neurons in the PBNL. n = number of mice (indicated in the graph). Error bars, s.e.m. Source data
Extended Data Fig. 3 Tacr1+ and Gpr83+ spinal PNs that innervate the posterior thalamus, midbrain or pons are distinct populations.
a, d, g, Schematics of lumbar spinal cord injections of AAV1–Con/Fon–EYFP viruses and brain injections of AAV2–retro–FlpO viruses into the SCig of Tacr1CreERT2 mice (a) (n = 3 mice), the MGm/SPFp of Tacr1CreERT2 (d) (n = 2 mice) or Gpr83CreERT2 mice (g) (n = 3 mice). b, e, h, Transverse sections of cervical spinal cords of Tacr1CreERT2 (b, e) or Gpr83CreERT2 mice (h). White dotted lines, tdTomato-expressing axons travelling through spinal cord white matter. DLF, dorsal lateral funiculus; VLF, ventral lateral funiculus. c, f, i, Coronal sections of target brain regions of Tacr1+ (c, f) or Gpr83+ (i) spinal PNs. AQ, cerebral aqueduct.
Extended Data Fig. 4 Strong axon terminal stimulation of Tacr1+ and Gpr83+ SPB neurons produces distinct locomotor behaviours.
a, Association of synaptic terminals of Tacr1+ and Gpr83+ SPB neurons with Calca-GFP-expressing cell bodies and neurites in the PBNEL. b, Quantification of the number of synaptophysin–tdTomato puncta associated with GFP+ cell bodies and neurites. The numbers were normalized with the total GFP+ area (to normalize for the variability of total GFP+ area) and the total number of synaptophysin-tdTomato puncta within the entire PBNL (to normalize for the variability of virus injections). AU, arbitrary unit. Two-tailed t-test; n = 4 mice each for Tacr1+ and Gpr83+ SPB neurons. c, Quantification of average speed during light-off periods following light-on periods (473 nm, 6.5 mW, 10 ms pulse width). One-way ANOVA (Dunnett’s multiple-comparisons test); F2,16 = 10.60 (2 Hz), F2,16 = 40.12 (5 Hz), F2,16 = 20.48 (10 Hz). d, Average velocity of mice over time (6.5 mW, 2 Hz, 10 ms pulse width). Positive values indicate forward movement whereas negative values indicate backward movement. Shaded areas, s.e.m. e, f, Quantification of average velocity during light-on periods with 2 Hz (e) and 5 Hz (f) photostimulation. Note that mice receiving Tacr1+ SPB neuron terminal stimulation exhibited net negative velocity during the 2 Hz photostimulation and lack a velocity increase despite the dramatic increase in speed during 5 Hz photostimulation. Two-tailed t-test; n = 6, 5 mice for Tacr1+, Gpr83+, respectively. g, Distribution of Fos+ neurons in the spinal cord dorsal horn following either photostimulation of axon terminals of SPB neurons (Tacr1+ or Gpr83+) or a capsaicin (0.1%) injection into a hindpaw. Photostimulation of axon terminals of SPB neurons did not induce significant Fos expression in the spinal cord, whereas a hindpaw injection of capsaicin induced strong Fos expression in the medial region of the superficial lamina of the spinal cord dorsal horn. d, dorsal; v, ventral, m, medial; l, lateral. n = 4, 3, 5, 2 mice for control, Gpr83+, Tacr1+, capsaicin, respectively. h, Quantification of the number of Fos+ neurons in laminae I and II. The number of Fos+ cells was quantified in the medial 200 μm of the spinal cord dorsal horn. One-way ANOVA (Tukey’s multiple-comparisons test). Error bars, s.e.m. Source data
a, b, Summary violin plots of peak instantaneous firing rates of Gpr83+ (a) and Tacr1+ (b) SPB neurons in response to von Frey indentations and thermal stimuli. Red lines indicate median, while blue lines indicate quartiles. Friedman test (Dunn’s multiple-comparisons test). n = 16, 15 neurons for Tacr1+, Gpr83+, respectively. c, Representative traces of action potential firing evoked by topical capsaicin (0.05%) treatment. Arrows, time when capsaicin was applied to the skin. d, Quantification of peak instantaneous firing rates upon capsaicin application. Mann–Whitney test (two-tailed); P value is indicated; n = 11, 7 neurons for Tacr1+, Gpr83+, respectively; error bars, s.e.m. Source data
Extended Data Fig. 6 Simultaneous inhibition of the synaptic outputs of both Tacr1+ and Gpr83+ SPB neurons attenuates nocifensive behaviours in response to noxious cutaneous stimuli.
a, Hindpaw-licking was scored while Tacr1CreERT2; Lbx1FlpO; Rosa26LSL-FSF-TeTx mice, Gpr83CreERT2; Lbx1FlpO; Rosa26LSL-FSF-TeTx mice or Tacr1CreERT2; Gpr83CreERT2; Lbx1FlpO; Rosa26LSL-FSF-TeTx mice were placed on the 55 °C hot plate (cut-off time, 20 s). These intersectional strategies target the entire Tacr1+ and Gpr83+ spinal populations, of which 34.2% (20.5% PBNL-projecting, 6.6% PAG-projecting and 7.1% MGm/SPFp-projecting PNs are combined) and 30.9% (14.0% PBNL-projecting, 4.6% PAG-projecting and 12.3% MGm/SPFp-projecting PNs are combined) are Tacr1+ and Gpr83+ PNs (laminae I and IIo and the LSN are combined), respectively (a detailed description of the quantification is in the methods). Two-tailed t-test. b, Forepaw licking was scored while mice were placed on the 5 °C cold plate (cut-off time, 3 min). Two-tailed t-test. c, Paw withdrawal frequency following hindpaw skin indentation using von Frey filaments. Two-way ANOVA; P value is indicated; F1,43 = 8.65 for Tacr1/Gpr83-TeTx. d, Real-time texture aversion assay (150 grit sand paper vs 400 grit sand paper). % of time spent in rough side of sand paper (150 grit) was measured (normalized to baseline preference). Two-tailed t-test. e, f, The suppression of neurotransmission in the quadruple transgenic mice was confirmed by reduced Fos induction in the PBNL following exposure of mice to noxious thermal stimuli. e, Distribution of Fos+ neurons in the PBNL following thermal stimulation. f, Quantification of the number of Fos+ neurons in the PBNL. One-way ANOVA (Tukey’s multiple-comparisons test); F2,9 = 8.97 (5 °C), F2,8 = 27.09 (55 °C). n = number of mice (indicated in the graphs). Error bars, s.e.m. Source data
Extended Data Fig. 7 Gpr83+ and Tacr1+ SPB neurons receive strong synaptic inputs from Mrgprd+ polymodal non-peptidergic sensory neurons and weak, sparse, and polysynaptic inputs from distinct primary sensory neurons, and exhibit distinct dendritic morphologies.
a, Distribution of CGRP+, Mrgprb4+, Mrgprd+ and Ntrk2+ primary afferent synaptic terminals in the spinal cord dorsal horn. The Rosa26FSF-LSL-SYN-GFP reporter mouse line33 was used in combination with sensory neuron Cre/FlpE mouse lines and AvilFlpO/AvilCre mouse lines. Note that CGRP+, Mrgprb4+, Mrgprd+ and Ntrk2+ primary afferent synaptic terminals mainly innervate laminae I and IIo, IIid, IIid, and IIiv and III, respectively. b–d, Quantifications of peak current density in Tacr1+ (c, d) and Gpr83+ (b) SPB neurons elicited by long light pulse-stimulation (1 ms and 10 ms) of CGRP+ (b), Mrgprb4+ (c) and Ntrk2+ (d) primary afferent terminals. The same neurons, stimulated with different durations of light stimulation, are connected by dotted lines. Note that only a small fraction of Gpr83+ SPB neurons exhibited long-latency (21.68 ± 2.66 ms), high-jitter (2.97 ± 0.85 ms) polysynaptic EPSCs with 10-ms-long photostimulation of CGRP+ afferent terminals and, conversely, only a small fraction of Tacr1+ SPB neurons exhibited long-latency (14.29 ± 3.49 ms), high-jitter (4.31 ± 2.31 ms) polysynaptic EPSCs with 10-ms-long photostimulation of Mrgprb4+ afferent terminals. 2 out of 7 Tacr1+ SPB neurons exhibited long-latency (11.89 ± 4.18 ms) but relatively low-jitter (0.57 ± 0.21 ms) synaptic EPSCs after 10-ms-long photostimulation of Ntrk2+ afferent terminals. e, Representative traces of light-activated currents (left) and AP firing (right) upon photostimulation of Mrgprd+ primary afferent terminals. Turquoise bars, 0.1 ms (EPSCs) and 1 ms (APs) LED (473 nm) stimulations. f, Quantifications of peak current density. Mann–Whitney test (two-tailed); n = number of neurons. g, Schematic of injections of AAV2-retro-FlpO viruses into the PBNL. h, Distribution of tdTomato-expressing dendrites of Tacr1+ (top) and Gpr83+ (bottom) SPB neurons. Lamina IIid is labelled using IB4 binding. Arrowheads, Gpr83+ dendrites that are extended into deeper laminae of the spinal cord dorsal horn. i, Quantification of distance between the cell bodies and the outer boundary of IB4+ lamina IIid (dotted line). #, note that a small number of Gpr83+ SPB neurons have their cell bodies located within lamina IIid. n = 65, 60 neurons for Tacr1+, Gpr83+, respectively. j, k, Quantifications of total length of dendrites in a spinal cord section image within (j) or below (k) IB4+ lamina IIid (normalized to the total length of the IB4+ lamina IIid in the same spinal cord section image). Two-tailed t-test; n = 18, 23 sections (40 μm) for Tacr1+, Gpr83+, respectively. Error bars, s.e.m. Source data
Extended Data Fig. 8 Anatomical analyses of axonal projections of anterolateral pathway PNs innervating the PBNL and the inferior olivary complex.
a, Schematic of dual-CTB injections into the PBNL. b, Distribution of CTB-labelled neurons in the spinal cord laminae I and IIo and the LSN. c, Quantification of % of SPB neurons that innervate the PBNL contralaterally, ipsilaterally or bilaterally. n = 3 mice. Error bars, s.e.m. d, Bottom view of a single axon trace of sparsely labelled Gpr83+ spinal PN that innervate the inferior olivary complex. Arrowhead, an axon branch travelling up to the rostral brain. r, rostral; c, caudal, m, medial; l, lateral. e, Quantification of the number of inferior olivary complex-projecting spinal PNs that exhibit dedicated vs. collateral-forming axons. f, Synaptic terminals of Tacr1+ (left) or Gpr83+ (right) PNs, representing hindlimb regions (GFP) and forelimb regions (tdTomato), are segregated in the inferior olivary complex. n = 3 mice each for Tacr1+ and Gpr83+ PNs. Source data
Extended Data Fig. 9 Photostimulation of either Tacr1+ or Gpr83+ SPB neuron axon terminals promotes rostral grooming, and produces distinct behaviours in instrumental conditioning assays.
a, Duration of rostral grooming of control (black line), Gpr83CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR (green line) or Tacr1CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR (red line) mice over time. Bin size, 30 s. Axon terminals in the PBNL were stimulated with blue LED (473 nm, 1 mW, 10 Hz, 10 ms pulse width) for 30 s 4 times (with 1-min light-off periods between photostimulation periods). Turquoise bars, 30-s-long light-on periods. b, Quantification of average duration of rostral grooming during light-on periods for 0.4 mW, 1 mW, and 6.5 mW photostimulation. One-way ANOVA (Dunnett’s multiple-comparisons test); F2,18 = 7.60 (1 mW), F2,16 = 7.49 (6.5 mW); n = 6, 6, 9 mice (0.4 mW), 6, 7, 9 mice (1 mW), 8, 5, 6 mice (6.5 mW) for control, Gpr83, Tacr1, respectively. c, Schematic of lumbar injections of AAV1–hSyn–FlpO viruses. d, Quantification of total duration of grooming of different body parts during light-on periods. Axon terminals in the PBNL were stimulated with blue LED (473 nm, 10 mW, 5 or 10 Hz, 10 ms pulse width) 4 times for 1 min each (with 1-min light-off periods between photostimulation periods). n = 4 trials (2 mice; 2 trials per mouse, 5 Hz and 10 Hz stimulation) for Tacr1+ SPB neuron terminal stimulation, n = 6 trials (3 mice; 2 trials per mouse, 5Hz and 10 Hz stimulation) for Gpr83+ SPB neuron terminal stimulation. Paired t-test (two-tailed). e, Weak self-administered photostimulation (0.4 mW) of Gpr83+ SPB neuron terminals led to an increase in the number of presses for the active lever, but not the inactive lever over time. f, Self-administered photostimulation (1 mW) of Tacr1+ SPB neurons led to a decrease in the number of presses for the active lever, but not inactive lever over time. Turquoise boxes indicate 8 d of light-on sessions. n = 7 mice (Gpr83, 0.4 mW; Tacr1, 1 mW). Two-way repeated measures ANOVA; F1,6 = 8.23 (Gpr83, 0.4 mW), F1,6 = 9.43 (Tacr1, 1 mW). Error bars, s.e.m. Source data
Extended Data Fig. 10 Summary of two parallel ascending SPB pathways and a phylogenetic tree of structurally-related GPCR family proteins.
a, Summary cartoon of two parallel ascending SPB pathways for affective touch and pain. b, A phylogenetic tree generated using a multiple sequence alignment algorithm, ClustalW2 (EMBL-EBI). The top 14 mouse proteins that have the highest amino acid sequence similarity to mouse GPR83 were used for this analysis.
Supplementary Table 1. p-values for statistical comparisons. Exact p-values for each statistical comparison.
Strong activation of Gpr83+ SPB terminals induces forward locomotion. A video of a Gpr83CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR mouse receiving high-intensity photostimulation (6.5 mW, 5 Hz, 10 ms pulse width).
Strong activation of Tacr1+ SPB terminals induces backward locomotion. A video of a Tacr1CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR mouse receiving high-intensity photostimulation (6.5 mW, 5 Hz, 10 ms pulse width).
Strong activation of Tacr1+ SPB terminals induces jumping. A video of a Tacr1CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR mouse receiving high-intensity photostimulation (6.5 mW, 10 Hz, 10 ms pulse width).
Freezing behavior is induced during light-off periods, following strong activation of either Gpr83+ or Tacr1+ SPB terminals. A video of a Gpr83CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR mouse after receiving high-intensity photostimulation (6.5 mW, 5Hz, 10 ms pulse width).
Activation of Tacr1+ SPB terminals induces rostral grooming. A video of a Tacr1CreERT2; Lbx1FlpO; Rosa26LSL-FSF-ReaChR mouse receiving moderate-intensity photostimulation (1 mW, 10Hz, 10 ms pulse width).
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Choi, S., Hachisuka, J., Brett, M.A. et al. Parallel ascending spinal pathways for affective touch and pain. Nature 587, 258–263 (2020). https://doi.org/10.1038/s41586-020-2860-1
Science Advances (2021)
Neuroscience Bulletin (2021)