Distinct Roles of Dopamine Receptor Subtypes in the Nucleus Accumbens during Itch Signal Processing

Abstract

Ventral tegmental area (VTA) dopaminergic neurons, which are well known for their central roles in reward and motivation-related behaviors, have been shown to participate in itch processing via their projection to the nucleus accumbens (NAc). However, the functional roles of different dopamine receptor subtypes in subregions of the NAc during itch processing remain unknown. With pharmacological approaches, we found that the blockade of dopamine D1 receptors (D1R), but not dopamine D2 receptors (D2R), in the lateral shell (LaSh) of the NAc impaired pruritogen-induced scratching behavior in male mice. In contrast, pharmacological activation of D2R in both the LaSh and medial shell (MeSh) of the NAc attenuated the scratching behavior induced by pruritogens. Consistently, we found that dopamine release, as detected by a dopamine sensor, was elevated in the LaSh rather than the MeSh of the NAc at the onset of scratching behavior. Furthermore, the elevation of dopamine release in the LaSh of the NAc persisted even though itch-relieving behavior was blocked, suggesting that the dopamine signal in the NAc LaSh represents a motivational component of itch processing. Our study revealed different dynamics of dopamine release that target neurons expressing two dopamine receptors subtypes within different subregions of the NAc, and emphasized that D1R in the LaSh of the NAc is important in itch signal processing.SIGNIFICANCE STATEMENT Dopamine has been implicated in itch signal processing. However, the mechanism underlying the functional role of dopamine in itch processing remains largely unknown. Here, we examined the role of dopamine D1 receptor (D1R) and D2R in the nucleus accumbens (NAc) shell during pruritogen-induced scratching behavior. We demonstrated that D1R in the NAc lateral shell (LaSh) play an important role in motivating itch-induced scratching behavior, while activation of D2R would terminate scratching behavior. Our study revealed the diverse functional roles of dopamine signals in the NAc shell during itch processing.

Keywords: dopamine receptors; itch; nucleus accumbens.

Figure 1.

Effects of manipulation of D1R or D2R in the NAc LaSh on scratching behavior. A, Left panel, Schematic of cannula implantation in the NAc LaSh. Right panel, Histologic verification of the location of cannulas in an example animal; arrows indicate the outline of cannulas. Scale bar, 500 μm. B, Timeline of the antagonist injection and scratching behavior test. C, Schematic of the scratching behavior recording chamber. D, Example trace showing the recorded scratching behavior. E, Raster plot showing CQ-induced scratching bouts in the three groups of mice after drug injection. Each bar represents a single scratching bout (n = 6–7 mice in each group). SCH23390, the antagonist of D1R; raclopride, the antagonist of D2R. F, Effects of dopamine receptor antagonists on CQ-induced scratching behavior. Left panel, Number of CQ-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site). Right panel, Total number of CQ-induced scratching bouts within 30 min of injection of saline, SCH23390, or raclopride (one-way ANOVA, n = 6–7 mice in each group, SCH23390 p = 0.008, raclopride p = 0.82). G, Effects of dopamine receptor antagonists on ET-1-induced scratching behavior. Left panel, Number of ET-1-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl site). Right panel, Total number of ET-1-induced scratching bouts within 30 min of injection of saline, SCH23390, or raclopride (one-way ANOVA, n = 5–7 mice in each group, SCH23390 p = 0.01, raclopride p > 0.99). H, Locomotion traces of mice that received injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site) in the open field test. I, Traveling distance in the open field test of mice in the three groups. Left panel, Traveling distance of mice after every 5 min. Right panel, Total traveling distance of mice in three groups (one-way ANOVA, n = 6–7 mice in each group). J, Diagram of a mouse with an adhesive sticker. K, Effect of dopamine receptor antagonists on sticker-induced scratching behavior. Left panel, Number of sticker-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site). Right panel, Total number of sticker-induced scratching bouts within 30 min of injection of saline, SCH23390, or raclopride (one-way ANOVA, n = 6–7 mice in each group). L, Effects of dopamine receptor agonists on CQ-induced scratching behavior. Left panel, Number of CQ-induced scratching bouts every 5 min after injection of saline, SKF38393 (8 μg/μl, 0.5 μl per site), or quinpirole (4 μg/μl, 0.5 μl per site). Right panel, Total number of CQ-induced scratching bouts within 30 min of injection of saline, SKF38393, or quinpirole (n = 7 mice per group, one-way ANOVA, SKF38393 p > 0.99, quinpirole p = 0.003). M, Traveling distance in the open field test of mice in the three groups. Left panel, Traveling distance of mice after every 5 min. Right panel, Total traveling distance of mice in three groups (n = 7 mice per group, one-way ANOVA, SKF38393 p = 0.24, quinpirole p = 0.88). N, Effects of dopamine D2 receptor agonist on sticker-induced scratching behavior. Left panel, Number of sticker-induced scratching bouts every 5 min after injection of saline or quinpirole (4 μg/μl, 0.5 μl per site). Right panel, Total number of sticker-induced scratching bouts within 30 min of injection of saline or quinpirole (n = 4–5 mice in each group, Mann-Whitney test, p = 0.03). O, The locations of the tips of injection tubes in mice. Each red circle represents an injection tube (n = 40 mice). All error bars represent SEM; *p < 0.05, **p < 0.01.

Figure 2.

Effects of manipulation of D1R or D2R in the NAc MeSh on scratching behavior. A, Left panel, Schematic of cannula implantation in the NAc MeSh. Right panel, Histologic verification of the location of the cannulas in an example animal; arrows indicate the outline of cannulas. Scale bar, 500 μm. B, Timeline of the antagonist injection and scratching behavior test. C, Raster plot showing CQ-induced scratching bouts detected in three groups of mice after drug injection. Each bar represents a single scratching bout (n = 7–8 mice in each group). D, Effects of dopamine receptor antagonists on CQ-induced scratching behavior. Left panel, Number of CQ-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site). Right panel, Total number of CQ-induced scratching bouts within 30 min after injection of saline, SCH23390, or raclopride (n = 7–8 mice in each group, one-way ANOVA, SCH23390 p = 0.71, raclopride p = 0.92). E, Effects of dopamine receptor antagonists on ET-1-induced scratching behavior. Left panel, Number of ET-1-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site), Right panel, Total number of ET-1-induced scratching bouts within 30 min after injection of saline, SCH23390, or raclopride (n = 8 mice per group, one-way ANOVA, SCH23390 p > 0.99, raclopride p > 0.99). F, Locomotion traces of mice that received an injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site) in the open field test. G, Traveling distance in the open field test of mice in the three groups. Left panel, Traveling distance of mice every 5 min. Right panel, Total traveling distance of mice in the three groups (n = 7–8 mice in each group, one-way ANOVA, SCH23390 p = 0.09, raclopride p = 0.51). H, Effects of dopamine receptor antagonists on sticker-induced scratching behavior. Left panel, Number of sticker-induced scratching bouts every 5 min after injection of saline, SCH23390 (0.2 μg/μl, 0.5 μl per site), or raclopride (0.2 μg/μl, 0.5 μl per site). Right panel, Total number of sticker-induced scratching bouts within 30 min after injection of saline, SCH23390, or raclopride (n = 8 mice per group, one-way ANOVA, SCH23390 p > 0.99, raclopride p > 0.99). I, Effects of dopamine receptor agonists on CQ-induced scratching behavior. Left panel, Number of CQ-induced scratching bouts every 5 min after injection of saline, SKF38393 (8 μg/μl, 0.5 μl per site), or quinpirole (4 μg/μl, 0.5 μl per site). Right panel, Total number of CQ-induced scratching bouts within 30 min after injection of saline, SKF38393, or quinpirole (n = 7–8 mice in each group, one-way ANOVA, SKF38393 p = 0.64, quinpirole p = 0.02). J, Traveling distance in the open field test of the three groups. Left panel, Traveling distance of mice every 5 min. Right panel, Total traveling distance of mice in three groups (n = 6–8 mice in each group, one-way ANOVA, SKF38393 p = 0.85, quinpirole p > 0.99). K, Effects of dopamine receptor agonists on sticker-induced scratching behavior. Left panel, Number of sticker-induced scratching bouts every 5 min after injection of saline, SKF38393 (8 μg/μl, 0.5 μl per site), or quinpirole (4 μg/μl, 0.5 μl per site). Right panel, Total number of sticker-induced scratching bouts within 30 min after injection of saline, SKF38393, or quinpirole (n = 8 mice per group, one-way ANOVA, SKF38393 p = 0.53, quinpirole p < 0.001). L, The locations of the tips of injection tubes in mice. Each red circle represents an injection tube (n = 24 mice). All error bars represent SEM; *p < 0.05, ***p < 0.001.

Figure 3.

Dopamine release in the NAc LaSh increased during itch-induced scratching behavior. A, Schematic of virus injection and optic fiber implantation in the NAc LaSh. B, Histologic verification of the location of the optic fiber in an example NAc LaSh^D1-DA2h animal. The arrow indicates the outline of the optic fiber. Scale bar, 500 μm. C, Schematic of simultaneous recording of the fluorescence signals of DA2h by fiber photometry during scratching behavior. D, Left panel, Representative DA2h fluorescence trace in NAc LaSh D1R⁺ neurons (top) and behavioral trace (bottom) in response to an intradermal injection of CQ. Right panel, Representative DA2h fluorescence trace in NAc LaSh D2R⁺ neurons (top) and behavioral trace (bottom) in response to an intradermal injection of CQ. Yellow shading indicates a scratching train. E, Dopamine signal detected by D1R⁺ neurons in the NAc LaSh at the onset of the scratching train in an example mouse in response to intradermal injection of CQ. Left panel, Individual scratching train trace aligned to the train onset of an example mouse. Middle panel, Heatmap of the DA2h fluorescence signal during each corresponding scratching train of an example mouse. Right panel, Average fluorescence change of all mice, with shaded area indicating SEM (n = 11 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the baseline (B.s.) period (B.s., −4 to −2 s) and the scratching train onset period (Onset, 0.5–1.5 s) used to quantify the fluorescent signal. F, Dopamine signal detected by D1R⁺ neurons in the NAc LaSh during scratching offset in the CQ model. Left panel, Scratching train trace of an example mouse aligned to the scratching train offset. Middle panel, Heatmap of the DA2h fluorescence signal during each scratching train of the same mouse. Right panel, Average DA2h fluorescence change of the whole group of mice, with shaded area indicating SEM (n = 11 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the scratch train offset period (Offset, 0.25–1.25 s) used to quantify the fluorescence. G, Dopamine signal detected by D2R⁺ neurons in the NAc LaSh at the onset of the scratching train in an example mouse in response to intradermal injection of ET-1. Left panel, Individual scratching train trace aligned to the train onset of an example mouse. Middle panel, Heatmap of the DA2h fluorescence signal during each corresponding scratching train of an example mouse. Right panel, Average fluorescence change of all mice, with shaded area indicating SEM (n = 20 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the baseline period (B.s., −4 to −2 s) and the scratching train onset period (Onset, 0.5–1.5 s) used to quantify the fluorescent signal. H, Dopamine signal detected by D2R⁺ neurons in the NAc LaSh during scratching offset in the CQ model. Left panel, Scratching train trace of an example mouse aligned to the scratching train offset. Middle panel, Heatmap of the DA2h fluorescence signal during each scratching train of the same mouse. Right panel, Average DA2h fluorescence change of the whole group of mice, with shaded area indicating SEM (n = 20 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the scratching train offset period (Offset, 0.25–1.25 s) used to quantify the fluorescence. I, Quantification of the average DA2h fluorescence change in NAc LaSh D1R⁺ neurons during the onset of scratching in the CQ (left panel) and ET-1 (right panel) models (two-way ANOVA, in the CQ model, p < 0.001 at onset, p = 0.60 at offset; in the ET-1 model, p < 0.001 at onset, p = 0.97 at offset). J, Quantification of the average DA2h fluorescence change in NAc LaSh D2R⁺ neurons during the onset of scratching in the CQ (left panel) and ET-1 (right panel) models (two-way ANOVA, in CQ model, p < 0.001 at onset, p = 0.12 at offset; in ET-1 model, p = 0.04 at onset, p = 0.28 at offset). All error bars represent SEM; *p < 0.05, ***p < 0.001. K, The locations of the tips of optic fibers in recorded mice. Each red circle represents an individual animal (D1-Cre n = 11 mice, D2-Cre n = 20 mice).

Figure 4.

Diverse dynamics of dopamine release in the NAc MeSh during itch-induced scratching behavior. A, Schematic of virus injection and optic fiber implantation in the NAc MeSh. B, Histologic verification of the location of the optic fiber in an example NAc MeSh^D1-DA2h animal. The arrow indicates the outline of the optic fiber. Scale bar, 500 μm. C, Schematic of simultaneous recording of the fluorescence signals of DA2h by fiber photometry during scratching behavior. D, Left panel, Representative DA2h fluorescence trace in NAc MeSh D1R⁺ neurons (top) and behavioral trace (bottom) in response to an intradermal injection of CQ. Right panel, Representative DA2h fluorescence trace in NAc MeSh D2R⁺ neurons (top) and behavioral trace (bottom) in response to an intradermal injection of CQ. Yellow shading indicates a scratching train. E, Dopamine signal detected by D1R⁺ neurons in the NAc MeSh at the onset of the scratching train in an example mouse. Left panel, Individual scratching train trace aligned with the train onset of an example mouse. Middle panel, DA2h fluorescence signal during each corresponding scratching train of an example mouse. Right panel, Average fluorescence change of all mice, with shaded area indicating SEM (n = 6 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the baseline period (B.s., −4 to −2 s) and the scratching train onset period (Onset, 0.5–1.5 s) used to quantify the fluorescent signal. F, Dopamine signal detected by D1R⁺ neurons in the NAc MeSh during scratching offset in the CQ model. Left panel, Scratching train trace of an example mouse aligned to the scratching train offset. Middle panel, Heatmap of the DA2h fluorescence signal during each scratching train of the same mouse. Right panel, Average DA2h fluorescence change of the whole group of mice, with shaded area indicating SEM (n = 6 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the scratching train offset period (Offset, 0.25–1.25 s) used to quantify the fluorescence. G, Dopamine signal detected by D2R⁺ neurons in the NAc MeSh at the onset of the scratching train in an example mouse. Left panel, Individual scratching train trace aligned to the train onset of an example mouse. Middle panel, DA2h fluorescence signal during each corresponding scratching train of an example mouse. Right panel, Average fluorescence change of all mice, with shaded area indicating SEM (n = 6 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the baseline period (B.s., −4 to −2 s) and the scratching train onset period (Onset, 0.5–1.5 s) used to quantify the fluorescent signal. H, Dopamine signal detected by D2R⁺ neurons in the NAc MeSh during scratching offset in the CQ model. Left panel, Scratching train trace of an example mouse aligned to the scratching train offset. Middle panel, Heatmap of the DA2h fluorescence signal during each scratching train of the same mouse. Right panel, Average DA2h fluorescence change of the whole group of mice, with shaded area indicating SEM (n = 6 mice in DA2h group, n = 4 mice in eGFP group). The black bar indicates the scratching train offset period (Offset, 0.25–1.25 s) used to quantify the fluorescence. I, Quantification of the average DA2h fluorescence change in NAc MeSh D1R⁺ neurons during the onset of scratching in the CQ (left panel) and ET-1 (right panel) models (two-way ANOVA, in the CQ model, p = 0.09 at onset, p = 0.99 at offset; in the ET-1 model, p = 0.48 at onset, p = 0.86 at offset). J, Quantification of the average DA2h fluorescence change in NAc MeSh D2R⁺ neurons during the onset of scratching in the CQ (left panel) and ET-1 (right panel) models (two-way ANOVA, in CQ model, p = 0.56 at onset, p = 0.003 at offset; in ET-1 model, p = 0.06 at onset, p = 0.004 at offset). All error bars represent SEM. K, The locations of the tips of optic fibers in recorded mice. Each red circle represents an individual animal (D1-Cre n = 6 mice, D2-Cre n = 6 mice); **p < 0.01.

Figure 5.

Optostimulation of cervical or lumbar spinal GRPR⁺ neurons induced scratching or turning and biting behavior. A, Schematic of virus injection in the cervical spinal cord. B, Histologic verification of the expression of the virus encoding ChR2-mCherry in the cervical spinal cord. Scale bar, 200 μm. C, Scratching behavior induced by activation of cervical spinal GRPR⁺ neurons. D, The probability of successful induction of scratching behavior by different intensities of optogenetic activation of cervical GRPR⁺ neurons (n = 10 mice). E, Optogenetic stimulation of cervical spinal GRPR^ChR2 neurons induced intensity-dependent scratching behavior. F, Optogenetic stimulation of cervical spinal GRPR^mCherry neurons failed to evoke scratching behavior. G, Histologic verification of the expression of the virus encoding ChR2-mCherry in the lumbar spinal cord. Scale bar, 200 μm. H, Biting behavior induced by activation of lumbar spinal GRPR⁺ neurons. I, The probability of turning or biting behavior induced by different intensities of optogenetic activation of lumbar spinal GRPR⁺ neurons (n = 13 mice). J, Probability of mice turning and biting while wearing collars of different sizes (two-way ANOVA, p < 0.001); ***p < 0.001.

Figure 6.

Dopamine release to D1R⁺ neurons in the NAc LaSh did not represent the reward signal accompanied by itch relief. A, Schematic of virus injection in the lumbar spinal cord (left panel) and virus injection and optic fiber implantation in the NAc LaSh (right panel). B, Left panel, Histologic verification of virus expression in the lumbar spinal cord (red: ChR2, blue: DAPI; scale bar, 200 μm). Right panel, Virus expression and optic fiber location in the NAc LaSh. The arrow indicates the outline of the fiber (green: DA2h; scale bar, 500 μm). C–E, Dopamine signals near D1R⁺ neurons in the NAc LaSh during activation of lumbar spinal GRPR⁺ neurons at 5 Hz (C), 10 Hz (D), and 20 Hz (E) optogenetic stimulation in an example mouse. Top panel, Average DA2h dopamine fluorescence signal during each trial of spinal GRPR⁺ neuron activation, with the shaded area indicating the SEM. Bottom panel, Heatmap of each trial. F, Top panel, Average dopamine signal at different intensities aligned to the onset of response behavior (turning), with the shaded area indicating SEM. Bottom panel, Average dopamine signal at different intensities aligned to the onset of itch relief (biting), with the shaded area indicating SEM (n = 7 mice in the GRPR^ChR2 group, n = 5 mice in the GRPR^mCherry group). G, Diagram of a mouse wearing a small collar during fiber photometry recording. Collar could not block biting or turning behaviors. H, Left panel, Average DA2h fluorescence change of an example mouse wearing a small collar during lumbar spinal GRPR⁺ neuron activation at 20 Hz, with the shaded area indicating SEM. Right panel, Each trial of the example mouse wearing a small collar during spinal GRPR⁺ neuron activation at 20 Hz. I, Average dopamine fluorescence change of mice wearing small collars, with shaded area indicating SEM (n = 7 mice). The green bar indicates the baseline period (B.s., −1 to 0 s), and the orange bar indicates the scratch train onset period (Onset, 0.25–0.75 s) that was used to quantify the fluorescent signal. J, Quantification of the dopamine fluorescence signal in mice wearing small collars (Wilcoxon test, p = 0.03). K, Diagram of a mouse wearing a normal collar during fiber photometry recording. L, Left panel, Average DA2h fluorescence change of an example mouse wearing a normal collar during lumbar spinal GRPR⁺ neuron activation at 20 Hz, with the shaded area indicating SEM. Right panel, Each trial of the example mouse wearing a normal collar during spinal GRPR⁺ neuron activation at 20 Hz. M, Average dopamine fluorescence change of mice wearing normal collars, with shaded area indicating SEM (n = 7 mice in GRPR^ChR2 group, n = 5 mice in GRPR^mCherry group). The green bar indicates the baseline period (B.s., −1 to 0 s), and the orange bar indicates the scratching train onset period (Onset, 0.25–0.75 s) that was used to quantify the fluorescent signal. N, Quantification of dopamine fluorescence signals in mice wearing normal collars (Wilcoxon test, p = 0.02). O, Quantification of dopamine fluorescence signals in the early (0.25–0.75 s) and late (1.25–1.75 s) phases during activation of GRPR⁺ neurons of mice wearing small collars and normal collars (two-way ANOVA; B.s., p > 0.999; First, p = 0.12; Last, p = 0.02). P, The locations of the tips of optic fibers in the recorded mice. Each red circle represents an individual animal (n = 7 mice). All error bars represent SEM; *p < 0.05.