Extended Data Fig. 4: Manipulation of descending LC-NAergic pathway and assessments for Hes5⁺ astrocyte-specific α_1A-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-hM3Dq^FLEX 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-hM3Dq^FLEX 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 α_1A-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 α_1A-AR in Hes5-CreERT2;AAV-shScramble^FLEX (n = 292 cells, 9 slices, 3 mice) and shAdra1a^FLEX 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