Innocuous warming enhances peripheral serotonergic itch signaling and evokes enhanced responses in serotonin-responsive dorsal horn neurons in the mouse

Abstract

Itch is often triggered by warming the skin in patients with itchy dermatitis, but the underlying mechanism is largely unknown. We presently investigated if warming the skin enhances histamine- or serotonin (5-HT)-evoked itch behavior or responses of sensory dorsal root ganglion (DRG) cells, and if responses of superficial dorsal horn neurons to innocuous warming are enhanced by these pruritogens. In a temperature-controlled environmental chamber, mice exhibited greater scratching following intradermal injection of 5-HT, but not histamine, SLIGRL, or BAM8-22, when the skin surface temperature was above 36°C. Calcium imaging of DRG cells in a temperature-controlled bath revealed that responses to 5-HT, but not histamine, were significantly greater at a bath temperature of 35°C vs. lower temperatures. Single-unit recordings revealed a subpopulation of superficial dorsal horn neurons responsive to intradermal injection of 5-HT. Of these, 58% responded to innocuous skin warming (37°C) prior to intradermal injection of 5-HT, while 100% responded to warming following intradermal injection of 5-HT. Warming-evoked responses were superimposed on the 5-HT-evoked elevation in firing and were significantly larger compared with responses pre-5-HT, as long as 30 min after the intradermal injection of 5-HT. Five-HT-insensitive units, and units that either did or did not respond to intradermal histamine, did not exhibit any increase in the incidence of warmth sensitivity or in the mean response to warming following intradermal injection of the pruritogen. The results suggest that 5-HT-evoked responses of pruriceptors are enhanced during skin warming, leading to increased firing of 5-HT-sensitive dorsal horn neurons that signal nonhistaminergic itch.

New & noteworthy: Skin warming often exacerbates itch in patients with itchy dermatitis. We demonstrate that warming the skin enhanced serotonin-evoked, but not histamine-evoked, itch behavior and responses of sensory dorsal root ganglion cells. Moreover, serotonin, but not histamine, enhanced responses of superficial dorsal horn neurons to innocuous warming. The results suggest that skin warming selectively enhances the responses of serotonin-sensitive pruriceptors, leading to increased firing of serotonin-sensitive dorsal horn neurons that signal nonhistaminergic itch.

Keywords: 5-HT; histamine; itch; mice; pain; pruritus; spinal neurons; warming.

Fig. 1.

Scratching elicited by 5-HT is enhanced under conditions of innocuous warming. Five-HT (10 μg/10 μl) or histamine (50 μg/10 μl) (A) and SLIGRL (50 μg/10 μl) or BAM8-22 (100 μg/10 μl) (B) were injected intradermally into the rostral back of mice. Mice were then placed into an environmental chamber where the temperature was set at 23, 25, or 28°C. Skin surface temperature of rostral back (gray triangle) was measured by thermocouple. The mean skin surface temperature was significantly higher at the 28°C chamber temperature (*P < 0.01, one-way ANOVA). The mean number of histamine-, SLIGRL-, or BAM8-22-evoked scratch bouts did not significantly vary with chamber temperature, while the mean number of 5-HT-evoked scratch bouts was significantly greater at 28°C (*P < 0.05, one-way ANOVA, n = 6/group).

Fig. 2.

Responses of DRG cells to 5-HT are enhanced by innocuous warming (35°C). A: representative examples of two DRG cells' responses to 5-HT (100 μM, 30 sec), followed by high potassium solution (HiK) at the indicated bath temperatures. Response was considerably larger at 35°C. B: representative examples of two DRG cells' responses to histamine (100 μM, 30 sec) at the indicated bath temperatures. Responses at both temperatures were of similar magnitude. C: mean responses of DRG cells to 5-HT (black square) or histamine (gray circle) at each tested temperature. Each data point is a separate group of DRG cells (see Table 1). Peak responses to 5-HT were significantly enhanced at 35°C relative to those at other lower temperatures (*P < 0.05, one-way ANOVA). In contrast, mean peak responses to histamine did not significantly differ across temperatures.

Fig. 3.

Enhancement of warming-evoked response of dorsal horn neuron after 5-HT. Peristimulus-time histogram (bins: 1 sec) showing, from left to right, cotton brush and pinch stimuli, warming stimulus, intradermal injection of saline, intradermal injection of 5-HT, and warming stimuli. Bars show time of warming stimuli. Top left inset: mechanosensitive receptive field (gray) on hindpaw.

Fig. 4.

Enhancement of warming-evoked responses in 5-HT-sensitive dorsal horn neurons. A: 5-HT-sensitive units (n = 12). Bar graphs indicate mean peak responses to warming stimulus (open column, number of impulses) and mean unit firing rate over 30 sec just prior to the warming stimulus (closed column, impulses per second) vs. time following intradermal injection of 5-HT (at time 0). Pre-5-HT responses are shown at the Pre time point. Error bars: SE. *Significant difference compared with corresponding responses before 5-HT (P < 0.05; paired t-test). B: peristimulus-time histogram (bins: 1 sec) of mean responses to warming stimulus before 5-HT. Inset: histologically recovered recording sites (dots) compiled on representative lumbar section. C: as in B for 5 min after 5-HT. D: as in B for 30 min after 5-HT.

Fig. 5.

Warming-evoked responses in 5-HT-insensitive dorsal horn neurons. A: 5-HT-insensitive units (n = 36). Bar graphs indicate mean peak responses to warming stimulus (open column) and mean unit firing rate over a 30-s period just prior to the warming stimulus (closed column) vs. time following intradermal injection of 5-HT (at time 0). Pre-5-HT responses are shown at the Pre time point. Error bars: SE. B: peristimulus-time histogram (bins: 1 sec) of mean responses to warming stimulus before 5-HT. Inset: histologically recovered recording sites (dots) compiled on representative lumbar section. C: as in B for 5 min after 5-HT. D: as in B for 30 min after 5-HT.

Fig. 6.

Individual example showing warming-evoked response of dorsal horn neuron before and after histamine. Peristimulus-time histogram (bins: 1 sec) showing, from left to right, cotton brush and pinch stimuli, warming stimulus, intradermal injection of saline, intradermal histamine, and warming stimuli. Bars show time of warming stimuli. Top middle inset: mechanosensitive receptive field (gray) on hindpaw.

Fig. 7.

Warming-evoked responses in histamine-sensitive dorsal horn neurons. A: histamine-sensitive units (n = 15). Bar graphs indicate mean peak responses to warming stimulus (open column) and mean unit firing rate over a 30-s period just prior to the warming stimulus (closed column) vs. time following intradermal injection of histamine (at time 0). Prehistamine responses are shown at the Pre time point. Error bars: SE. *Significant difference compared with corresponding responses before histamine (P < 0.05; paired t-test). B: peristimulus-time histogram (bins: 1 sec) of mean responses to warming stimulus before histamine. Inset: histologically recovered recording sites (dots) compiled on representative lumbar section. C: as in B for 5 min after histamine. D: as in B for 30 min after histamine.

Fig. 8.

Warming-evoked responses in histamine-insensitive dorsal horn neurons. A. histamine-insensitive units (n = 14). Bar graphs indicate mean peak responses to warming stimulus (open column) and mean unit firing rate over a 30-s period just prior to the warming stimulus (closed column) vs. time following intradermal injection of histamine (at time 0). Prehistamine responses are shown at the Pre time point. Error bars: SE. B: peristimulus-time histogram (bins: 1 sec) of mean responses to warming stimulus before histamine. Inset: histologically recovered recording sites (dots) compiled on representative lumbar section. C: as in B for 5 min after histamine. D: as in B for 30 min after histamine.