Ghrelin receptor antagonism of hyperlocomotion in cocaine-sensitized mice requires βarrestin-2

Abstract

The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through βarrestin-2 to regulate actin/stress fiber rearrangement, suggesting βarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific βarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The βarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for βarrestin-2 and show that pharmacological inhibition of βarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of βarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate βarrestin-2 activity differentially from G protein activity may be required.

Keywords: YIL781; addiction; arrestin; cocaine; ghrelin.

Figure 1. Effects of YIL781 on in cellulo signaling of the GHSR1a determined by G-protein mediated Ca²⁺ responsiveness and βarrestin-2 translocation

(A–B) Ca²⁺ response of the GHSR1a in HEK-293 cells was assessed by bioluminescence in the presence of an aequorin reporter. (A) YIL781 treatment alone over the range of concentrations was modeled by a line with slope = 0 ± 0.0078 and intercept = 0.47 ± 0.0077 (B) The ability of the GHSR1a agonist L-692,585 to activate calcium was inhibited by increasing doses of YIL781, IC₅₀= (1.85 ± 0.093) × 10⁻⁷ M). (C–D) βarrestin-2 translocation to the activated GHSR1a in U2OS cells was measured by assessing the formation of receptor/ βarrestin-2 aggregates using a robotic microscope plate reader. (C) Linear regression of YIL781 treatment over the dose range, Intercept = 18 ± 43, slope = 0.35 ± 6.2. (D) The ability of L-692,585 to induce βarrestin-2 translocation was inhibited by YIL781 with an IC₅₀= (1 ± 0.26) × 10⁻⁶ M. Data were analyzed by GraphPad Prism, version 7.02 and are presented as mean ± sem, N = 3 independent experiments.

Figure 2. Acute cocaine-induced locomotion after GHSR1a antagonist treatment

(A) Cocaine-induced locomotion was measured in C57BL/6J YIL781 treated WT mice (n=5–6 mice/group; ANOVA: interaction P < 0.0001; treatment P < 0.01). Tukey`s post hoc comparisons of different doses of YIL781 and vehicle revealed significant differences at multiple time points (*p<0.05). (B) Effect of pre-treatment with 10 and 20 mg/kg YIL781 on cocaine-mediated locomotion evaluated by total distanced moved over the 30 min post-cocaine injection period (ANOVA: treatment P < 0.0006) Tukey`s post hoc test: 10mg/kg YIL781 vs saline, *p< 0.05; 20mg/kg YIL781 vs saline, ***p < 0.001; 20mg/kg YIL781 vs 5 or 10 mg/kg YIL781, #p<0.05). (C) ANOVA analysis of 10 mg/kg YIL781 treatment on locomotor activity compared to vehicle (treatment P=0.66), interaction P=0.29).

Figure 3. Locomotor sensitization to cocaine in C57BL/6J mice

(A) On the test day, the GHSR1a antagonist pre-treatment reduced cocaine locomotion in both saline sensitized and cocaine sensitized groups at multiple time points (n= 6 mice/group, ANOVA: interaction P < 0.0001; treatment P< 0.0001; Tukey`s post hoc test: cocaine-sensitized group compared to saline sensitized group, #p<0.05; YIL781 compared to saline pre-treatment, *p<0.05). (B) Data showing the sum of distance traveled during 30 min after cocaine administration (ANOVA: treatment P < 0.0001; Tukey`s post hoc test: cocaine-sensitized group compared to saline sensitized group, #p<0.05, ##p<0.01; YIL781 compared to saline pre-treatment, *p<0.05, **p<0.01).

Figure 4. Blood pressure and heart rate in WT mice treated with YIL781

(A, B) Treatment with different doses of the GHSR1a antagonist YIL781 did not change either blood pressure (n=3, ANOVA for treatment P=0.60) or heart rate (n=3, ANOVA for treatment P=0.80) compared to vehicle treatment in WT mice.

Figure 5. Locomotor sensitization to cocaine in DAβarrestin-2 KO mice

(A, B) Representative images showing Cre-expression patterns (green) in midbrain dopamine neurons (DA) (magenta) in the two different DAT-Cre mouse lines. Scale bars = 100 µm. (C, D) Plots of 20 mg/kg cocaine induced locomotion for DAβarr2KO-1 and DAβarr2KO-2 mice on sensitization Day 1. Differences in the areas under the respective curves were observed for the grey shaded regions, (grey shaded area; n= 8–9 mice/group, for DAβarr2KO-1: p <0.0001; for DAβarr2KO-2: p <0.0001). (E, F) Cocaine-mediated locomotion on Day 5 of sensitization. (G, H) On the test day (Day 10), 10 mg/kg YIL781 pre-treatment reduced 5 mg/kg cocaine-induced locomotion in sensitized WT mice only (ANOVA: DAβarr2KO-1 interaction P < 0.0001; treatment P = 0.099; DAβarr2KO-2 interaction P < 0.0001; treatment P < 0.0001; Post hoc comparison: WT/YIL781 compared to WT/saline, *p<0.05; WT/YIL781 compared to DAβarr2KO-1 or DAβarr2KO--2/YIL781, #p<0.05). (I, J) Total distance traveled 30 min after cocaine administration. (ANOVA: DAβarr2KO-1 treatment P < 0.004; DAβarr2KO-2 treatment P < 0.005; post hoc test: WT/YIL781 compared to WT/saline, *p<0.05; WT/YIL781 compared to DAβarr2KO-1 or DAβarr2KO-2/YIL781, #p<0.05).

Figure 6. Locomotor sensitization to cocaine in whole body βarrestin-2 KO mice

(A) Plot of 20 mg/kg cocaine-induced locomotion for whole body βarr2KO mice and littermate WT control mice on sensitization Day 1. Differences in the areas under the respective curves were observed for the grey shaded regions (n = 24 mice/genotype, t = 2.623, p <0.05). (B) Cocaine-mediated locomotion on Day 5 of sensitization. (C) On the test day (Day 10), 10 mg/kg YIL781 pre-treatment reduced 5 mg/kg cocaine-induced locomotion in sensitized WT mice only. A two-way ANOVA revealed significant main effects for time (n = 11–12 mice/group, F (23, 989) = 64.5, p<0.0001) and treatment/genotype group (F (3, 43) = 3.499, p<0.05). Tukey’s post hoc comparison: WT/Vehicle compared to WT/YIL781, *p<0.05; βarr2KO/Vehicle compared to WT/YIL781, #p<0.05; WT/ YIL781 compared to βarr2KO/YIL781, $p<0.05. (D) Total distance traveled 30 min after cocaine administration. A one-way ANOVA identified a treatment group effect, F (3, 42) = 2.958, p < 0.05. Tukey’s post hoc comparison: WT/Vehicle compared to WT/YIL781, *p<0.05.

Figure 7. Schematic model for G protein and βarrestin-mediated GHSR1a signaling

(A) Individual G protein- and βarrestin-mediated signaling pathways of activated GHSR1a. (B) Conceptual model for GHSR1a regulation of VTA dopamine neurons underlying locomotion. Activation of βarrestin (red arrows) leads to N-methyl-D-aspartic acid receptor (NMDAR) enhanced excitability of VTA dopamine neurons, dopamine release, and locomotion. Inhibition of this pathway with a GHSR1a antagonist reduces dopamine neuron firing, dopamine release and locomotion, blocking the hyper-locomotor effect of psychostimulants like cocaine. (C) Model of the effect of a genetic ablation of βarrestin-2 in DAT neurons. DAT neurons in WT mice are still responsive to GHSR1a antagonists (upper panel, green sigmoid curve) and the negative change in locomotion, Δ locomotion = Δ locomotion (βarrestin, other factors) is a significant fraction of the total locomotion. In the YIL781 unresponsive βarrestin-2 KO mouse, (lower panel), Δ locomotion at the GHSR1a is fixed at zero because of compensation by other remaining regulatory mechanisms (lower panel, green sigmoid curve).