Oxytocin Elicits Itch Scratching Behavior via Spinal GRP/GRPR System

Abstract

Oxytocin (OT), a neuropeptide involved in the regulation of complex social and sexual behavior in mammals, has been proposed as a treatment for a number of psychiatric disorders including pain. It has been well documented that central administration of OT elicits strong scratching and grooming behaviors in rodents. However, these behaviors were only described as symptoms, few studies have investigated their underlying neural mechanisms. Thus, we readdressed this question and undertook an analysis of spinal circuits underlying OT-induced scratching behavior in the present study. We demonstrated that intrathecal OT induced robust but transient hindpaw scratching behaviors by activating spinal OT receptors (OTRs). Combining the pre-clinical and clinical evidence, we speculated that OT-induced scratching may be an itch symptom. Further RNAscope studies revealed that near 80% spinal GRP neurons expressed OTRs. OT activated the expression of c-fos mRNA in spinal GRP neurons. Chemical ablation of GRPR neurons significantly reduced intrathecal OT-induced scratching behaviors. Given GRP/GRPR pathway plays an important role in spinal itch transmission, we proposed that OT binds to the OTRs expressed on the GRP neurons, and activates GRP/GRPR pathway to trigger itch-scratching behaviors in mice. These findings provide novel evidence relevant for advancing understanding of OT-induced behavioral changes, which will be important for the development of OT-based drugs to treat a variety of psychiatric disorders.

Keywords: gastrin-releasing peptide; hindpaw scratching; itch; oxytocin; oxytocin receptor; spinal dorsal horn.

FIGURE 1

Intrathecal OT induced hindpaw scratching behavior was mediated by spinal OTRs. (A) Time course of different doses of intrathecal OT evoked scratching bouts in mice. OT at dose of 0.003, 0.01, 0.03, 0.1, and 0.3 nmol was administered intrathecally, scratching bouts of the mice were then recorded every 5 min for 30 min. (B) Different doses of intrathecal OT-induced scratching bouts in 30 min after injection. Data are expressed as mean ± SEM. **p < 0.01, ****p < 0.0001 vs. Saline; One-way ANOVA followed by Bonferroni’s post hoc test. (C) Dose-response curve of intrathecal OT-induced scratching behaviors. (D) Intrathecal OT-induced itch behavior in male and female mice. OT at dose of 0.03 nmol was administered intrathecally, scratching bouts was then recorded for 30 min. Data are expressed as mean ± SEM paired T- test. (E) Intrathecal administration of a selective OT receptor agonist, TGOT (0.03 nmol) caused scratching behaviors similar to OT in mice. (F) Intrathecal administration of selective V1aR antagonist, also a selective OT receptor agonist, TC OT 39 (0.1 nmol) caused significant scratching behaviors in mice. (G) Intrathecal administration of selective OT receptor antagonists, Atosiban (0.1 nmol) and dVOT (0.1 nmol) decreased OT induced scratching behaviors in mice. (H) Selective blockade of V1aR by pretreatment of its antagonist, RG7314 (0.1 nmol) did not prevent OT-induced scratching behaviors in mice. I Morphine (0.3 nmol, i.t.) had no effect on OT-induced scratching. Data are expressed as mean ± SEM paired T- test. *p < 0.05.

FIGURE 2

Co-expression of Otr mRNA and Grp mRNA in spinal dosal horn. (A) In situ hybridization for Otr and Grp mRNA on spinal dorsal horn sections. Co-expression of Otr mRNA (white) and Grp mRNA (green) in the enlarged image. Arrowheads indicate the co-expression of Otr and Grp mRNA. n = 9–15 sections from 3 mice. (B) percentage of neurons expressed Otr, Grp, or Otr-Grp mRNA. Data are expressed as mean ± SEM. (C) In situ hybridization for Otr and Grpr mRNA on spinal dorsal horn sections. Co-expression of Otr mRNA (white) and Grpr mRNA (pink) in the enlarged image. Arrowheads indicate the co-expression of Otr and Grpr mRNA. n = 9–15 sections from 3 mice. (D) percentage of neurons expressed Otr, Grpr, or Otr-Grpr mRNA. Data are expressed as mean ± SEM.

FIGURE 3

Ablation of GRPR neurons reduced OT-induced scratching behaviors in mice. (A) RNAscope showed Bombesin-saporin selectively ablated spinal GRP neurons. (B) Ablation of GRPR neurons reduced OT-induced scratching behaviors in mice. Mice were given a single intrathecal injection of Bombesin-saporin (400 ng). Intrathal OT-induced scratching bouts was then recorded for 30 min two weeks after Bombesin-saporin injection. Data are expressed as mean ± SEM. Student T- test. (C) Co-expression of Grp mRNA and c-fos mRNA in spinal dorsal horn. In situ hybridization for Grp (green) and c-fos (white) mRNA on spinal dorsal horn sections. Arrows indicate the co-expression of Grp and c-fos mRNA. n = 9–15 sections from 3 mice. (D) Schematic illustration of proposed spinal neural circuit that transmit OT-induced itch scratching behavior in mice.

FIGURE 4

Systemic OT failed to evoke scratching behaviors in mice. (A) Intradermal injection of OT failed to evoke scratching behaviors in mice. OT was administered intradermally, scratching bouts were then recorded for 30 min. Data are expressed as mean ± SEM. Student t-test. (B) The nuclei (blue) were counterstained with Dapi, and cell body of the neurons (white) was shown by Nissl staining. RNAScope data showed that Otr mRNA (red puncta) was not expressed in the DRG neurons. N = 9–15 sections from 3 mice. Scale bar, 100 μm.