Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity

Abstract

Astrocytes are critical regulators of CNS function and are proposed to be heterogeneous in the developing brain and spinal cord. Here we identify a population of astrocytes located in the superficial laminae of the spinal dorsal horn (SDH) in adults that is genetically defined by Hes5. In vivo imaging revealed that noxious stimulation by intraplantar capsaicin injection activated Hes5+ SDH astrocytes via α1A-adrenoceptors (α1A-ARs) through descending noradrenergic signaling from the locus coeruleus. Intrathecal norepinephrine induced mechanical pain hypersensitivity via α1A-ARs in Hes5+ astrocytes, and chemogenetic stimulation of Hes5+ SDH astrocytes was sufficient to produce the hypersensitivity. Furthermore, capsaicin-induced mechanical hypersensitivity was prevented by the inhibition of descending locus coeruleus–noradrenergic signaling onto Hes5+ astrocytes. Moreover, in a model of chronic pain, α1A-ARs in Hes5+ astrocytes were critical regulators for determining an analgesic effect of duloxetine. Our findings identify a superficial SDH-selective astrocyte population that gates descending noradrenergic control of mechanosensory behavior.

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Fig. 1: Identification of Hes5+ cells as a population of SDH astrocytes in adult mice.
Fig. 2: Ca2+ increases in supSDH astrocytes by noxious stimuli via descending NAergic signaling.
Fig. 3: Activation of the descending NAergic pathway elicits astrocytic [Ca2+]i increases in the supSDH.
Fig. 4: Activation of α1A-ARs in Hes5+ astrocytes induces mechanical hypersensitivity.
Fig. 5: Chemogenetic stimulation of Hes5+ SDH astrocytes induces mechanical hypersensitivity.
Fig. 6: d-serine is involved in Hes5+ SDH astrocyte-mediated mechanical hypersensitivity.
Fig. 7: Hes5+ SDH astrocytes contribute to capsaicin-induced hypersensitivity via descending NAergic signaling.
Fig. 8: Conditional α1A-AR knockout in Hes5+ astrocytes enhances the analgesic effect of duloxetine on neuropathic mechanical hypersensitivity.

Data availability

The data of the current study are presented in the figures. If necessary, the data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

No code was used for the study.

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Acknowledgements

We thank E. Sakaguchi for starting the initial experiment for rAAV, and the University of Pennsylvania vector core for providing pZac2.1, pAAV2/5, pAAV2/9 and pAd DeltaF6 plasmids. This work was supported by JSPS KAKENHI grant numbers JP19H05658, 19K22500 (to M.T.), JP16K18885, JP18K14821 (to Y.K.), JP25117013 (to K.I.), by the Core Research for Evolutional Science and Technology (CREST) program from the Japan Agency for Medical Research and Development (AMED) under grant number JP20gm0910006 (to M.T.), by the Practical Research Project for Allergic Diseases and Immunology (Research on Allergic Diseases and Immunology) from AMED under grant number JP17ek0410034 (to M.T.), the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant number JP20am0101091 (to M.T.), JP20am0101120 (to R. Kobayashi, T.H. and I.H.), by Naito Foundation (to Y.K. and M.T.), and by The Nakatomi Foundation (to M.T.). The authors appreciate Shiseido Co., Ltd., for their technical support for the measurement of d-serine. T.M. and K.Y. were research fellows of the JSPS (15J03522 and 19J21063, respectively).

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Authors

Contributions

Y.K. designed and performed most of the behavioral experiments, analyzed the data and wrote the manuscript. T.M. and K.Y. performed the in vivo imaging experiments, the Ca2+ imaging experiments using spinal cord slices, analyzed the data and wrote the manuscript. K. Kohno performed fluorescence in situ hybridization. K. Koga provided assistance for the experiments involving chemogenetic stimulation of LC neurons. R. Katsuragi and K. Momokino performed the behavioral assays and immunohistochemistry. T. Oka and S.M. performed the Ca2+ imaging in spinal cord slices. T.Y. performed immunohistochemistry of the brain. R.T. assisted with the experiments. S.O., A.F. and K. Hamase performed the d-serine measurements. T. Oti and H.S. performed the in situ experiments. K. Hayashida measured the NE levels in the SDH. R. Kobayashi, T.H. and I.H. generated Adra1aflox/flox mice. H.T.-S. assisted with the experiments and designed a custom chamber for in vivo spinal cord imaging. K. Mikoshiba contributed the role of IP3R2. V.T. provided Hes5-CreERT2 mice. K.I. supervised the project. M.T. conceived this project, designed experiments, supervised the overall project and wrote the manuscript. All the authors read and discussed the manuscript.

Corresponding author

Correspondence to Makoto Tsuda.

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Extended data

Extended Data Fig. 1 Distribution of Hes5+ cells in the SDH, trigeminal nucleus, dorsal root ganglia, sciatic nerve and brain.

a, Immunostaining of tdTomato+ cells by cell-type markers (NeuN for neurons, IBA1 for microglia and APC for oligodendrocytes) in L4-SDH sections of Hes5-CreERT2;Rosa-tdTomato mice. The data are representative of three independent experiments. b, Double immunostaining of SDH tdTomato+ cells by SOX9 and IB4 (a marker of non-peptidergic C fibres whose central terminals are located seletively in lamina IIi). Percentage of tdTomato+ astrocytes per total tdTomato+ cells and tdTomato+ astrocytes per total astrocytes (labeled by SOX9) in laminae I−IIi and III−V of Hes5-CreERT2;Rosa-tdTomato mice treated with tamoxifen during adulthood (right, n = 3 mice). c, tdTomato expression in the brainstem including the trigemical nucleus of Hes5-CreERT2;Rosa-tdTomato mice. d, Double immunostaining of tdTomato+ cells with SOX9 or NeuN in the spinal trigeminal nucleus caudalis (Sp5c) of Hes5-CreERT2;Rosa-tdTomato mice. e, Percentage of tdTomato+ cells with each marker in the Sp5c (n = 3 mice per group). f, g, tdTomato expression in a dorsal root ganglion (DRG, f) and sciatic nerve (g) of Hes5-CreERT2;Rosa-tdTomato mice. NeuN (f) and SOX10 (a marker for Schwann cells) (g) were used to counterstain DRG and sciatic nerve, respectively. h, i, tdTomato expression in the sagittal (h) and coronal (i) brain sections of Hes5-CreERT2;Rosa-tdTomato mice. jm, Double immunostaining of tdTomato+ cells for SOX9 (an astrocytic marker) in cortex (j), hippocampus (k), thalamus (l) and hypothalamus (m) of Hes5-CreERT2;Rosa-tdTomato mice. n, Double immunostaining of tdTomato+ cells for NeuN in cortex (left) and hippocampus (right). The data in fn were representative of three independent experiments. Data show the mean ± s.e.m. Scale bars, 20 μm (a, d), 100 μm (jn), 200 μm (b, f, g), 1 mm (c, i) and 2 mm (h).

Extended Data Fig. 2 supSDH astrocytic Ca2+ responses to several stimuli using in vivo imaging.

a, Schematic illustration of in vivo imaging of the superficial SDH in mice using two-photon microscopy. The chamber was attached at the L3−4 vertebral level. b, Immunohistochemical identification of GCaMP6m-expressing cells using cell-type markers (SOX9 and GFAP, NeuN, and IBA1). Note that GCaMP6m-expressing cells were immunolabeled by SOX9 and GFAP but not by NeuN or IBA1, indicating that GCaMP6m is expressed selectively in astrocytes. The data are representative of three independent experiments. c, Peak amplitude of astrocytic [Ca2+]i in the left SDH was high during the first 15 min (but not 55–60 min) after intraplantar injection of capsaicin (n = 59 ROIs, 3 mice). A von Frey filament (vF; 0.6 or 2.0 g) was applied to the left hindpaw before (pre) and 60 min after the injection. These light mechanical stimuli did not elicit astrocytic [Ca2+]i increases. Kruskal-Wallis test with Dunn’s multiple comparisons test. d, Representative Ca2+ traces in individual supSDH astrocytes in mice injected with capsaicin into the ipsilateral (left) and contralateral side (right). Synchronous and persistent Ca2+ responses were observed in the ipsilateral but not in the contralateral side. Data show the mean ± s.e.m. Scale bar, 20 µm. See source data for statistical parameters. Source data

Extended Data Fig. 3 Pharmacological responsiveness of the supSDH astrocytes to 5-HT and NE, and generation of Adra1aflox/flox mice.

a, Amplitudes of Ca2+ increases evoked by bath application of 5-HT (10 or 100 μM) and NE (10 μM) to spinal cord slices (n = 86 cells, 4 slices, 4 mice). b, Effect of adrenergic agonists (n = 68 cells, 3 slices, 3 mice for phenylephrine, n = 60 cells, 3 slices, 3 mice for clonidine, n = 64 cells, 3 slices, 3 mice for isoproterenol) on astrocytic Ca2+ levels. Note that the α1-AR agonist phenylephrine produced Ca2+ increases, but the α2-AR agonist clonidine and the β-AR agonist isoproterenol did not. Data show the mean ± s.e.m. c, Schematic illustration to generate a conditional allele at the Adra1a locus. d, Sequences of ssODNs with 5′- and 3′-homology arms flanking loxP and a restriction site. Asterisks indicates a phosphorothioate bond. e, Immunofluorescence of α1A-ARs in Adra1afloxflox and Hes5-CreERT2;Adra1afloxflox mice with tamoxifen administration. The data are representative of three independent experiments. Scale bar, 20 µm.

Extended Data Fig. 4 Manipulation of descending LC-NAergic pathway and assessments for Hes5+ astrocyte-specific α1A-AR-knockdown.

a, Co-expression of tyrosine hydroxylate (TH) immunoreactivity (green) and tdTomato expression in brainstem NAergic nuclei. To determine the main region of the descending NAergic pathway projecting to the SDH in mice, we injected the retrograde virus AAV2-retro expressing Cre under the control of the enhanced synapsin promoter (AAV2-retro-ESYN-Cre) in the SDH of Rosa-tdTomato mice. Note that a number of tdTomato+ LC-NAergic neurons were observed in the ventral part of the LC. The data are representative of three independent experiments. b, NE transporter immunofluorescence in the SDH 3 days after saline or DSP-4 treatments. Fluorescence intensity ratio of NE transporter in the SDH of saline or DSP-4 treated mice (n = 3 mice per group, two-tailed unpaired t-test). c, NE content in the SDH of saline or DSP-4 treated mice (n = 5 mice for saline, n = 6 mice for DSP-4; two-tailed unpaired t-test). d, Schematic illustration of experimental approach. Microinjection of AAV2-retro-ESYN-Cre into the SDH of wild-type mice, and subsequently, AAV-hM3DqFLEX into the bilateral LC. e, Immunolabeling of HA-tag (green) in the brainstem of mice injected with AAV2-retro-ESYN-Cre in to the SDH and AAV-hM3DqFLEX into the LC. f, Immunolabeling of HA-tag (green), mCherry (magenta) and TH (white) in the LC of mice transduced with hM3Dq into LC-NAergic neurons. In e and f, the images were representative of three independent experiments. g, Schematic timeline for tamoxifen treatments and behavioral experiments. Mice were injected with tamoxifen once a day for 10 days (days 12 to 21 after rAAV injection) and behavioral experiments (assessment of mechanical hypersensitivity after intrathecal injection of phenylephrine or intraplantar injection of capsaicin) were conducted 7 days after the last tamoxifen treatment. h, Amplitude of Ca2+ increases evoked by phenylephrine (0.3 μM) and ionomycin (1 μM) in primary cultured astrocytes (n = 23 cells for shScramble, n = 17 cells for shAdra1a; two-tailed Mann-Whitney test and two-tailed unpaired t-test). The shAdra1a suppressed the α1A-AR-mediated Ca2+ responses without affecting ionomycin-induced Ca2+ increases. i, Amplitude of SDH astrocytic Ca2+ increases evoked by phenylephrine (30 µM; n = 43 cells, 5 slices, 4 mice for shScramble, n = 47 cells, 5 slices, 4 mice for shAdra1a; two-tailed Mann-Whitney test). j, Immunofluorescence (left) and fluorescence intensity (right) of α1A-AR in Hes5-CreERT2;AAV-shScrambleFLEX (n = 292 cells, 9 slices, 3 mice) and shAdra1aFLEX mice (n = 270 cells, 9 slices, 3 mice). ROIs were manually selected based on DAPI (blue) /mCherry (magenta) signals (middle). Two-tailed Mann-Whitney test. Scale bars, 20 μm (j middle), 100 μm (j left), 200 μm (a, b, f) and 1 mm (e). Data show the mean ± s.e.m. See source data for statistical parameters. Source data

Extended Data Fig. 5 Functional expression of hM3Dq in Hes5+ SDH astrocytes.

a, Schematic illustration of the experimental protocol and timeline in Hes5-CreERT2 mice using the Cre-On system. AAV-hM3DqFLEX was microinjected unilaterally into the SDH of Hes5-CreERT2 mice. Tamoxifen treatment induced the expression of hM3Dq in Cre-positive (Hes5+) cells. b, Double immunostaining of hM3Dq (HA-tag) and cell-type markers (NeuN, IBA1 and APC) in the SDH of Hes5-CreERT2;AAV-hM3DqFLEX mice. The data are representative of three independent experiments. c, Representative images of the Ca2+ response (left) and an example of traces and average data (right) for the effect of CNO treatment on the fluorescence intensity of ROIs in wild-type and Hes5-CreERT2;AAV-hM3DqFLEX mice, both of which expressed GCaMP6m in SDH astrocytes. CNO induced astrocytic Ca2+ increases in spinal cord slices taken from Hes5-CreERT2;AAV-hM3DqFLEX mice but not from wild-type;AAV-hM3DqFLEX mice. The data are representative of three independent experiments. Scale bars, 20 μm (b) and 100 μm (c).

Extended Data Fig. 6 Chemogenetic stimulation of SDH astrocytes and Hes5-negative astrocytes in deeper laminae of the SDH.

a, Schematic illustration of the experimental protocol. AAV-hM3DqFLEX was microinjected unilaterally into the SDH of Gfap-Cre mice. b, Immunofluorescence of hM3Dq (HA-tag) in the SDH of Gfap-Cre and wild-type mice injected with AAV-hM3DqFLEX. Note that hM3Dq expression was induced only in Gfap-Cre;AAV-hM3DqFLEX mice and that its localization was observed in the SDH, including superficial laminae. c, Immunohistochemical identification of hM3Dq (HA-tag)-expressing cells using cell-type markers (SOX9, GFAP, NeuN, IBA1 and APC) in the SDH of Gfap-Cre;AAV-hM3DqFLEX mice. Arrows indicate hM3Dq-positive cells. hM3Dq was selectively expressed in astrocytes. The data in b and c were representative of three independent experiments. d, hM3Dq-induced mechanical hypersensitivity in Gfap-Cre;AAV-hM3DqFLEX mice (saline, n = 4 mice; CNO, n = 5 mice); repeated measures two-way ANOVA with Bonferroni’s multiple comparison test and Kruskal-Wallis test with Dunn’s multiple comparisons test. The letter indicates the following P value versus the ipsilateral side of the saline group (a, P < 0.0001). e, Schematic illustration of the experimental protocol and timeline in Hes5-CreERT2 mice using the Cre-Off system. AAV-gfaABC1D-hM3DqFLEX was microinjected unilaterally into the SDH of Hes5-CreERT2 mice, in which the hM3Dq sequence can be inverted by tamoxifen to stop hM3Dq expression in Cre-positive (Hes5+) cells. By contrast, Cre-negative (Hes5-negative) astrocytes (located in the deeper laminae) express hM3Dq. f, c-Fos+ neurons in laminae I–IIi and III–V of vehicle or CNO-treated Gfap-Cre;AAV-hM3DqFLEX mice with or without Aβ fiber stimulation (vehicle without stimuli, n = 6 mice; CNO without stimuli, n = 5 mice; vehicle with stimuli, n = 6 mice; CNO with stimuli, n = 6 mice; one-way ANOVA with Tukey’s multiple comparisons test). Insets: c-Fos immunofluorescence in laminae I–IIi. Scale bars, 50 μm (c), 100 μm (f insets) and 200 μm (b, f). Dashed lines (b, f) indicate the boundary between the gray and white matters. Data show the mean ± s.e.m. See source data for statistical parameters. Source data

Extended Data Fig. 7 Involvement of IP3R2 and D-serine signaling on NMDA receptors for astrogliogenic hypersensitivity.

a, Immunolabeling of IP3R2 in the SDH of wild-type and Ip3r2–/– mice. b, Immunohistochemical identification of IP3R2-expressing cells using cell-type markers (SOX9, GFAP, NeuN and APC) in the SDH of wild-type mice. Note that almost all IP3R2-positive cells in the SDH were astrocytes. The data in a and b were representative of three independent experiments. c, Representative images of Ca2+ response (left) and an example of traces and average data (right) for the effect of CNO (100 μM) treatment on the fluorescence intensity of ROIs in Gfap-Cre;Ip3r2–/–;AAV-hM3DqFLEX mice in which SDH astrocytes expressed GCaMP6m. CNO did not induce Ca2+ increases in Gfap-Cre;Ip3r2–/– mice. The data are representative of three independent experiments. d, PWT of Gfap-Cre;Ip3r2+/+ and Gfap-Cre;Ip3r2–/– mice injected with AAV-hM3DqFLEX before and after CNO administration (n = 4 mice per group; repeated measures two-way ANOVA with Bonferroni’s multiple comparisons test). The letters indicate the following P values versus the ipsilateral side of Gfap-Cre;Ip3r2+/+ mice (a, P = 0.0005; b, P < 0.0001; c, P = 0.0478). e, c-Fos+ neurons in laminae I–IIi and III–V of CNO-treated Gfap-Cre;Ip3r2–/–;AAV-hM3DqFLEX mice with (n = 6 mice) or without (n = 5 mice) Aβ fiber stimulation (two-tailed unpaired t-test). CNO did not increase the number of SDH neurons positive for c-Fos in laminae I–IIi and III-V. fj, Effect of forced expression of dnSNARE (f) and pharmacological blockade of receptors for NMDA (g), AMPA (h), ATP (i) and of a glycine site on NMDA receptors (j) on the CNO-induced mechanical hypersensitivity in Gfap-Cre;AAV-hM3DqFLEX mice (dnSNARE, MK-801, CNQX and DCK, n = 5 mice per group; PPADS, n = 4 mice per group); repeated measures two-way ANOVA with Bonferroni’s multiple comparisons test. The letters indicate the following P values versus the ipsilateral side of hM3Dq group (f: a, P = 0.0002), versus the ipsilateral side of CNO + Saline treated group (g: a, P = 0.0011), and versus the ipsilateral side of CNO + Vehicle treated group (j: a, P = 0.0002). k, PWL in wild-type mice 30 min after intrathecal injection of saline or D-serine (100 nmol) (n = 6 mice per group, two-tailed unpaired t-test). l, c-Fos+ neurons in laminae I–IIi and III–V of wild-type mice 30 min after intrathecal injection of D-serine (100 nmol) with (n = 5 mice) or without (n = 4 mice) Aβ fiber stimulation; two-tailed unpaired t-test. m, Schematic illustration of experimental protocol (left). Extracellular D-serine levels (% D-serine) in supernatant of spinal cord slices (taken from Hes5-CreERT2;AAV-hM3DqFLEX mice) 2 min after vehicle or CNO (100 μM) application (right, n = 11 mice per group, two-tailed unpaired t-test). Scale bar, 20 μm (b) and 100 μm (a, c, l). Dashed lines (a and l) indicate the boundary between the gray and white matters. Data show the mean ± s.e.m. See source data for statistical parameters. Source data

Extended Data Fig. 8 α1A-AR-knockdown of Hes5-negative astrocytes in deeper laminae of the SDH.

Schematic illustration of the Cre-OFF system to knockdown α1A-ARs in Cre-negative (Hes5-negative) astrocytes. AAV-gfaABC1D-shRNA (shScramble or shAdra1a)FLEX was microinjected unilaterally into the SDH of Hes5-CreERT2 mice. Assessment of mechanical hypersensitivity after intraplantar injection of capsaicin was conducted 7 days after the last tamoxifen treatment.

Extended Data Fig. 9 Distribution of Hes5+ SDH astrocytes after peripheral nerve injury.

a, Schematic timeline for tamoxifen treatments and fixation. Mice were injected with tamoxifen once a day for 5 days [days 5 to 9 or days 12 to 16 after peripheral nerve injury (PNI)] and were fixed 7 days after the last tamoxifen treatment. b, tdTomato expression in the SDH of Hes5-CreERT2;Rosa-tdTomato mice after PNI. c, Immunostaining of SOX9 in the ipsilateral SDH of Hes5-CreERT2;Rosa-tdTomato mice after PNI. Note that cell-type specificity and the expression pattern of tdTomato+ cells were not changed after PNI. In b and c, the data are representative of three independent experiments. Scale bars, 200 μm (c) and 500 μm (b).

Extended Data Fig. 10 Effect of intrathecal injection of NE and phenylephrine at higher doses on mechano- and thermosensory behaviors.

a, b, PWT (a) or PWL (b) in wild-type mice 30 min after intrathecal injection of noradrenaline (n = 6 mice per group; two-tailed paired t test and two-tailed Wilcoxon matched-pairs signed rank test). c, d, PWT (c) or PWL (d) in wild-type mice 30 min after injection of phenylephrine (n = 6 mice per group; two-tailed paired t test). Data show the mean ± s.e.m. See source data for statistical parameters. Source data

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Kohro, Y., Matsuda, T., Yoshihara, K. et al. Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-00713-4

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