Molecular and neural basis of contagious itch behavior in mice

Abstract

Socially contagious itch is ubiquitous in human society, but whether it exists in rodents is unclear. Using a behavioral paradigm that does not entail prior training or reward, we found that mice scratched after observing a conspecific scratching. Molecular mapping showed increased neuronal activity in the suprachiasmatic nucleus (SCN) of the hypothalamus of mice that displayed contagious scratching. Ablation of gastrin-releasing peptide receptor (GRPR) or GRPR neurons in the SCN abolished contagious scratching behavior, which was recapitulated by chemogenetic inhibition of SCN GRP neurons. Activation of SCN GRP/GRPR neurons evoked scratching behavior. These data demonstrate that GRP-GRPR signaling is necessary and sufficient for transmitting contagious itch information in the SCN. The findings may have implications for our understanding of neural circuits that control socially contagious behaviors.

Fig. 1. Mice display imitative scratching behavior

(A) Representative screen shots of a mouse observing a conspecific scratching in an adjacent home cage followed by imitative scratching. (B) Mean number of look behaviors toward the control or scratching demonstrator. (C) Mean number of instances of look-and-scratch (imitative scratch) behavior by the observers watching the scratching demonstrator versus those watching the control demonstrator. (D) Time course (in 5-min intervals) of mean number of imitative scratches. (E) Images of look-and-scratch behavior and screen shots of a mouse observing a video of a con-specific scratching followed by imitative scratching. (F) Mean number of looks toward the on-screen control or scratching demonstrators. (G) Mean number of imitative scratches by the observers watching the on-screen scratching demonstrator versus those watching the control demonstrator. (H) Time course of mean number of imitative scratches. n = 7 or 8 mice per group. Unpaired t test in (B), (C), (F), and (G); ***P < 0.001; ns, not significant. Data are means ± SEM.

Fig. 2. c-Fos and GRP expression in the suprachiasmatic nucleus of mice with contagious itch

(A) Heat map showing comparison of normalized fold change in number of c-Fos⁺ neurons in select brain regions between mice observing ambulating (control) or scratching demonstrators. (B) Schematic of coronal brain section with suprachiasmatic nucleus (SCN) enlarged with core and shell regions, third ventricle (3V), and optic chiasm (OC) indicated. (C and D) c-Fos expression in SCN of mice observing control (C) and scratching (D) demonstrators. (E) Mean number of c-Fos neurons in SCN of control and scratching observer groups. (F and G) GRP immunohistochemistry in SCN of mice observing control (F) and scratching (G) videos. (H) Mean normalized intensity of GRP staining in SCN sections of control and scratching observer groups. (I) Image of Grp in situ hybridization (ISH) in SCN. (J) Higher-magnification image of Grp in situ hybridization in the shell and core, respectively. (K and L) AVP immunohistochemistry image in SCN of mice observing control (K) and scratching (L) demonstrators. (M) Mean normalized intensity of AVP staining in SCN sections of control and scratching observer groups. Scale bars, 100 µm [(C), (D), (F), (G), (I), (K), and (L)], 20 µm (J). n = 3 mice and 10 sections per group. Unpaired t test in (E), (H), and (M); ***P < 0.001. Data are means ± SEM.

Fig. 3. The role of GRP-GRPR signaling in contagious itch

(A) Mean number of imitative scratches in Grp wild-type (WT) and KO or Grpr WT and KO littermates. (B) Mean number of looks in Grp WT and KO or Grpr WT and KO littermates. (C) Mean number of scratches induced by intradermal (i.d.) injection of histamine (200 µg) in Grp WT and KO or Grpr WT and KO littermates. (D) Schematic of targeting strategy for Grpr floxed allele. (E) Gel image of genotype polymerase chain reaction results from Grpr^wt/wt, Grpr^F/F, and Grpr^F/wt littermates. (F) Coronal brain section illustrating bilateral injection of AAV2-Syn-Cre-IRES-eGFP into SCN of Grpr^wt/y or Grpr^F/y mice. (G) Images of GRPR and eGFP expression in SCN of AAV2-Syn-Cre-eGFP–injected Grpr^wt/y or Grpr^F/y mice. Arrowheads indicate GRPR-eGFP double-positive neurons. (H) Mean number of GRPR⁺ neurons in SCN of AAV2-Syn-Cre-eGFP–injected Grpr^wt/y or Grpr^F/y mice. (I) Mean number of imitative scratches and mean number of looks in AAV2-Syn-Cre-eGFP–injected Grpr^wt/y or Grpr^F/y mice. Scale bars in (G), 100 µm (left column), 20 µm (other images). n = 7 mice per group for behavior; n = 3 mice and 14 to 16 sections per group for imaging. Unpaired t test in (A) to (C), (H), and (I); ***P < 0.001. Data are means ± SEM.

Fig. 4. Activation of GRP or GRPR neurons in the SCN evokes scratching behavior

(A) Mean number of scratches induced by microinjection of saline or GRP (0.04 nmol per 200 nl) into SCN of mice. (B) Time course of scratching induced by saline or GRP into SCN. (C) Image of c-Fos⁺ neurons in SCN after injection of saline or GRP. (D) Mean number of c-Fos neurons in SCN after injection of saline or GRP. (E) Brain sections illustrating unilateral injection of AAV2-Syn-DIO-ChR2-eYFP into SCN of Grp-Cre mice and subsequent fiber-optic implantation. (F) Images of GRP and ChR2-eYFP expression in SCN after viral injection. (G) Representative raster plots of scratching induced by photostimulation (473 nM for 2 min at 30 Hz) of eYFP control or ChR2 mice. (H) Mean number of scratches induced by photostimulation at 30 Hz in eYFP control mice or at 5, 10, 20, or 30 Hz in ChR2 mice. (I) Image of eYFP and c-Fos expression in SCN after photostimulation at 30 Hz in eYFP control or ChR2 mice. (J) Mean number of c-Fos⁺ neurons in SCN of eYFP control or ChR2 mice. (K) Diagram illustrating role of GRP-GRPR signaling in SCN for visually induced contagious itch. Scale bars, 50 µm [(C) and (I)], 100 µm [(F), far left], 20 µm [(F), far right]. n = 6 or 7 mice per group for behavior; n = 3 mice and 9 sections per group for imaging. Unpaired t test in (A), (D), and (J); one-way analysis of variance with Tukey post hoc in (H); **P < 0.01, ***P < 0.001. Data are means ± SEM.