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A central neural circuit for itch sensation

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Science  18 Aug 2017:
Vol. 357, Issue 6352, pp. 695-699
DOI: 10.1126/science.aaf4918
  • Fig. 1 Dissection of the spinoparabrachial pathway that mediates itch signal processing.

    (A) Schematic diagram for retrobeads injection and experimental timeline. (B and C) Representative images of c-Fos+ and beads+ neurons in the ipsilateral dorsal spinal cord after intradermal injection of (C) histamine or (B) saline. Arrowheads indicate double-labeled neurons. Scale bar, 50 μm. (D) Percentage of beads+ cells expressing c-Fos in the dorsal spinal cord (n = 6 or 7 mice). (E) Schematic diagram for intraspinal cord viral injection and optical fiber implantation in the PBN. (F) Effect of optogenetic inhibition of the spinoparabrachial pathway on scratching behavior in response to histamine. Each point represents the number of scratching bouts in a 3-min light on (593 nm, 8 to 10 mW, yellow shaded) or light off period (n = 16 or 17 mice). (G and H) The total number of scratching bouts during light on or off phase in response to histamine [(G), n = 16 or 17 mice] or chloroquine [(H), n = 16 or 17 mice]. (I) Schematic depicting virus and retrobeads injection, as well as recording configuration in spinal slices. (J) All recorded cells were filled with biocytin (blue) and were beads-positive (red). GRPR+ fibers were labeled with EYFP (green). Scale bar, 10 μm. (K) Action potentials induced through photostimulation (473 nm, 1 ms, blue bars) in spinal GRPR+ neurons. (L) Representative traces showing EPSCs evoked through photostimulation (473 nm, 1 ms) in a beads+ neuron in the spinal slice before and after NBQX (10 μM). (M) Summary data showing the amplitude of light-evoked EPSCs (n = 4 neurons), P = 0.056. Error bars represent SEM. *P < 0.05, ***P < 0.001. Unpaired t test for (D); one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons test for (G) and (H); paired t test for (M).

  • Fig. 2 Pharmacogenetic suppression of PBN neurons impaired itch-induced scratching behavior.

    (A) c-Fos expression in the PBN in response to histamine (right) or saline (left). Scale bar, 100 μm. (B and C) Number of c-Fos+ neurons in different parts of PBN in response to (B) histamine or (C) chloroquine as compared with control (n = 3 to 5 mice). Cont, contralateral; Ipsi, ipsilateral. (D) Schematic depicting the recording system for obtaining the Ca2+ signal with fiber photometry and scratching behavior with a magnetic induction method simultaneously. (E) Ca2+ transients associated with scratching behavior induced by histamine. (Top) The heatmap illustrating Ca2+ signals aligned to the beginning of scratching trains. Each row plots Ca2+ signals corresponding to one scratching train. Color scale indicates ΔF/F. (Bottom) Individual trial (light blue) and the averaged Ca2+ transients (red). (F and G) Mean fluorescent signal of mice injected with AAV-hSyn-GCaMP6s (red) or AAV-hSyn-EGFP (blue) in the PBN in response to histamine [(F), n = 5 or 7 mice] or chloroquine [(G), n = 5 or 6 mice], with shaded areas indicating SEM. (H) Expression of hM4Di-mCitrine or EGFP in the PBN. Scale bars, 200 μm (left), 25 μm (right). (I) (Top) hM4Di-mCitrine+ cells were recorded in cell-attached mode in acute brain slices. (Middle) Effect of bath application of CNO on spikes of an example hM4Di-mCitrine+ neuron in the PBN. (Bottom) Firing rate normalized to baseline (n = 4 neurons). (J) Timeline of the behavioral experiment. (K) Effect of pharmacogenetic inhibition of the PBN on the scratching behavior in response to histamine (n = 10 or 14 mice) or chloroquine (CQ) (n = 10 or 14 mice). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test for (B) and (C); one-way ANOVA with Bonferroni’s correction for multiple comparisons test for (K).

  • Fig. 3 Glutamatergic neurons in the PBN are required for itch-induced scratching behavior.

    (A) Bilateral injection of AAV-Cre-EGFP or AAV-EGFP into the PBN of Vglut2f/f mice. (B and C) In situ hybridization showing the reduction of Vglut2+ neurons in the PBN of Vglut2f/f mice that received AAV-Cre-EGFP injection compared with those that received AAV-EGFP injection (n = 5 or 8 mice). Scale bar, 150 μm. (D) The EPSCs induced through photostimulation (473 nm, 1 ms, blue bars) in amygdala of wild-type and Vglut2f/f mice injected with AAV-Cre-EGFP and AAV-DIO-ChR2-mCherry in the PBN (n = 12 or 13 neurons). Scale bars, 10 ms and 40 pA. (E) The number of scratching bouts in response to histamine in Vglut2f/f mice injected with AAV-EGFP or AAV-Cre-EGFP in the PBN (n = 10 or 14 mice). (F) Summary showing the effect of genetic deletion of VGLUT2 in the PBN on scratching behaviors induced with different pruritogens (n = 5 to 14 mice). (G) Number of scratching bouts in allergic itch model (n = 5 or 8 mice). (H) Experimental timeline of the DNFB model. (I and J) Number of scratching bouts in chronic itch model induced with DNFB at (I) 1 hour and (J) 24 hours after DNFB treatment (n = 10 or 11 mice). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t test for (C), (F), and (G); two-way ANOVA for (D), (I), and (J).

Supplementary Materials

  • A central neural circuit for itch sensation

    Di Mu, Juan Deng, Ke-Fei Liu, Zhen-Yu Wu, Yu-Feng Shi, Wei-Min Guo, Qun-Quan Mao, Xing-Jun Liu, Hui Li, Yan-Gang Sun

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S14 
    • References 

    Images, Video, and Other Media

    Movie S1
    Recording of scratching behavior with two methods.

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