Hemokinin-1 is a mediator of chronic restraint stress-induced pain

Abstract

The Tac4 gene-derived hemokinin-1 (HK-1) binds to the NK1 receptor, similarly to Substance P, and plays a role in acute stress reactions and pain transmission in mice. Here we investigated Tac4 mRNA expression in stress and pain-related regions and its involvement in chronic restraint stress-evoked behavioral changes and pain using Tac4 gene-deleted (Tac4^-/-) mice compared to C57Bl/6 wildtypes (WT). Tac4 mRNA was detected by in situ hybridization RNAscope technique. Touch sensitivity was assessed by esthesiometry, cold tolerance by paw withdrawal latency from 0°C water. Anxiety was evaluated in the light-dark box (LDB) and open field test (OFT), depression-like behavior in the tail suspension test (TST). Adrenal and thymus weights were measured at the end of the experiment. We found abundant Tac4 expression in the hypothalamic-pituitary-adrenal axis, but Tac4 mRNA was also detected in the hippocampus, amygdala, somatosensory and piriform cortices in mice, and in the frontal regions and the amygdala in humans. In Tac4^-/- mice of both sexes, stress-induced mechanical, but not cold hyperalgesia was significantly decreased compared to WTs. Stress-induced behavioral alterations were mild or absent in male WT animals, while significant changes of these parameters could be detected in females. Thymus weight decrease can be observed in both sexes. Higher baseline anxiety and depression-like behaviors were detected in male but not in female HK-1-deficient mice, highlighting the importance of investigating both sexes in preclinical studies. We provided the first evidence for the potent nociceptive and stress regulating effects of HK-1 in chronic restraint stress paradigm. Identification of its targets might open new perspectives for therapy of stress-induced pain.

Figure 1

Mouse Tac4 mRNA primarily expressed by excitatory neurons. Representative confocal images of Tac4 mRNA (red) co-localized with Vglut1 (green) and Gad1 mRNA (white) in the olfactory bulb (OB, Bregma 4 mm, (A), in the prelimbic cortex (PrL, Bregma –1.54 mm, (B), in the primary somatosensory cortex (S1, Bregma –1.46 mm, (C), in the CA1 region of hippocampus (CA1, Bregma –1.46 mm, (D), in the basolateral amygdala (BLA, Bregma –1.46 mm, (E), in the layer II of the piriform cortex (Pir, Bregma –1.46 mm, (F) and co-localized with Vglut2 (yellow) in the lateral periaqeductal gray (lPAG, Bregma –4.96 mm, (G) and in the raphe magnus nucleus (RMg, Bregma –4.96 mm, (H). Scale bar: 20 µm, inset scale bar: 10 µm.

Figure 2

Mouse Tac4 mRNA is expressed in the spinal cord and sensory neurons of trigeminal (TG) and dorsal root ganglia (DRG). Representative confocal images of Tac4 mRNA (red) in TG (A), in DRG (B) co-localized with (A) and without (B) calcitonin gene-related peptide (CGRP) mRNA (white). Tac4 was co-expressed with NeuN-positive (white) neurons of the Rexed Lamina II (C) and IV (D) of the lumbar (L4-L6) dorsal horn of the spinal cord. Scale bar: 20 µm, inset scale bar: 10 µm.

Figure 3

Mouse hypothalamic–pituitary–adrenal (HPA) axis and thymus expressed Tac4 mRNA. Representative confocal images of Tac4 mRNA (red) in the mouse HPA axis co-localized with corticotropin releasing hormone (Crh, green) of the paraventricular nucleus of the hypothalamus (PVN, Bregma –0.70 mm, (A), in the posterior and anterior lobe of the pituitary (C), in the adrenal cortex (D), and in the thymus (E). Brightfield microscopy image of the posterior and anterior pituitary gland (B). Scale bar: 20 µm, inset scale bar: 10 µm.

Figure 4

Mechanical hyperalgesia and cold allodynia in male and female WT and Tac4^−/− animals. Baseline mechanonociceptive thresholds in grams (A, E) and cold sensitivity in seconds (C, G) and their change in % after stress (B, D, F, H) during the 4-week-long experimental period. *p < 0.05, **p < 0.01, ***p < 0.001 represents the difference between WT and gene-deleted groups and ^#p < 0.05, ^###p < 0.001 represents the difference between respective non-stressed and stressed groups; for baseline values: t test, for changes: two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test.

Figure 5

Behavioral changes of WT and Tac4^−/− animals. Time spent in the center zone (A, D), latency to first entry to center zone (B, E) and body elongation time (C, F) in open field test at the end of the 4-week-long experimental period. *p < 0.05, **p < 0.01, ***p < 0.001 represents the difference between WT and gene-deleted groups and ^#p < 0.05, represents the difference between respective non-stressed and stressed groups; two-way analysis of variance (ANOVA) followed by Fischer’s post hoc test.

Figure 6

Behavioral changes in WT and Tac4^−/− animals. Immobility time in tail suspension test (A, D), time spent in the lit compartment (B, E) and transitions between the light and dark compartments in the light–dark box test (C, F) at the end of the 4-week-long experimental period. *p < 0.05, ***p < 0.001 represents the difference between WT and gene-deleted groups and ^#p < 0.05, ^##p < 0.01, ^##p < 0.001 represents the difference between respective non-stressed and stressed groups; two-way analysis of variance (ANOVA) followed by Fischer’s post hoc test.

Figure 7

Adrenal gland and thymus weights in WT and Tac4^−/− animals. Relative thymus weights (A, B), as well as relative adrenal gland weights (C, D) of the animals at the end of the 4-week-long experimental period. *p < 0.05, **p < 0.01 represents the difference between WT and gene-deleted groups and ^##p < 0.01, ^###p < 0.001 represents the difference between respective non-stressed and stressed groups; two-way analysis of variance (ANOVA) followed by Fischer’s post hoc test.