Orphan receptor-GPR52 inverse agonist efficacy in ameliorating chronic stress-related deficits in reward motivation and phasic accumbal dopamine activity in mice

Abstract

Reward processing dysfunctions e.g., anhedonia, apathy, are common in stress-related neuropsychiatric disorders including depression and schizophrenia, and there are currently no established therapies. One potential therapeutic approach is restoration of reward anticipation during appetitive behavior, deficits in which co-occur with attenuated nucleus accumbens (NAc) activity, possibly due to NAc inhibition of mesolimbic dopamine (DA) signaling. Targeting NAc regulation of ventral tegmental area (VTA) DA neuron responsiveness to reward cues could involve either the direct or indirect-via ventral pallidium (VP)-pathways. One candidate is the orphan G protein-coupled receptor GPR52, expressed by DA receptor 2 NAc neurons that project to VP. In mouse brain-slice preparations, GPR52 inverse agonist (GPR52-IA) attenuated evoked inhibitory postsynaptic currents at NAc-VP neurons, which could disinhibit VTA DA neurons. A mouse model in which chronic social stress leads to reduced reward learning and effortful motivation was applied to investigate GPR52-IA behavioral effects. Control and chronically stressed mice underwent a discriminative learning test of tone-appetitive behavior-sucrose reinforcement: stress reduced appetitive responding and discriminative learning, and these anticipatory behaviors were dose-dependently reinstated by GPR52-IA. The same mice then underwent an effortful motivation test of operant behavior-tone-sucrose reinforcement: stress reduced effortful motivation and GPR52-IA dose-dependently restored it. In a new cohort, GRAB_DA-sensor fibre photometry was used to measure NAc DA activity during the motivation test: in stressed mice, reduced motivation co-occurred with attenuated NAc DA activity specifically to the tone that signaled reinforcement of effortful behavior, and GPR52-IA ameliorated both deficits. These findings: (1) Demonstrate preclinical efficacy of GPR52 inverse agonism for stress-related deficits in reward anticipation during appetitive behavior. (2) Suggest that GPR52-dependent disinhibition of the NAc-VP-VTA-NAc circuit, leading to increased phasic NAc DA signaling of earned incentive stimuli, could account for these clinically relevant effects.

Fig. 1. Effect of GPR52 inverse agonist on inhibitory transmission at NAc-MSN to VP synapses.

A Schematic of the experimental design. AAV vector for ChR2 was injected into the NAc. Terminals of the infected MSNs (red) were activated with 470 nm light and responses were recorded in VP neurons. B Overlapped differential interference contrast and fluorescence image of a coronal slice (bregma 0.40 mm) in the recording chamber, with the recording electrode (shown by the arrow) placed at mCherry⁺ terminals in the VP. C Representative traces of IPSCs in VP neurons that were evoked by paired photo stimulation (blue bars, 50 ms inter-stimulus interval) of MSN terminals before (baseline) and after bath application of vehicle (individual traces in blue and average in black) or GPR52-IA (individual traces in red and average in black). Calibration bars represent 50 ms, 50 PA. D Time course of IPSC amplitude evoked by the photo stimulation of MSN terminals before and after bath application of GPR52-IA (red) or VEH (blue). Statistical analysis was conducted using linear mixed model with fixed effects of dose (D) and time (T) and a random effect of mouse subject. Significant interaction effects were analyzed using Sidak’s multiple comparisons test. *p < 0.05. E The area under the curve (AUC) of the time-course (0–40 min) of eIPSCs in VP neurons in GPR52-IA- and VEH-treated slices. **p = 0.004. Data shown are mean ± s.e.m. NAC nucleus accumbens, VP ventral pallidum, ac anterior commissure, Str striatum.

Fig. 2. Effects of chronic social stress and GPR52 inverse agonist on reward learning and effortful motivation.

A Experimental design. BBW + FC: measurement of baseline body weight and food consumption; 90–95% BBW/Conditioning: conditioning under food restriction that reduced BW to 90–95% BBW; CSS/CON: CSS protocol or control handling; Ad lib food re-BBW + FC: BW and food consumption under ad libitum feeding on days 5–12 of CSS/CON provided re-baseline values; 95–100% re-BBW: mice were mildly food restricted to be tested at 95–100% re-BBW. B CSS and CON protocols. C–G Discriminative reward learning-memory (DRLM) test. C Tone discriminative stimulus (DS) signaled chocolate pellet availability following a feeder response; maximum DS duration was 30 s per trial and inter-trial intervals (ITIs) were 20–80 s (mean = 50 s). Mice received 3 daily tests of 40 trials each and trials 1–30 per test were used for data analysis. Data are shown as mean ± SEM per test and individual mouse scores. Statistical analysis was conducted using linear mixed models with fixed effects of group (G), dose (D) and test (T) and a random effect of mouse subject. Significant main or interaction effects were analyzed using Sidak’s multiple comparisons test. *p < 0.05, ***p < 0.001. Test days indicated by different letters were significantly different (p < 0.05). D Number of chocolate pellets obtained i.e. DS trials with a response. E Median DS response latency. F Median ITI response interval. G Median learning ratio (ITI response interval/DS response latency. H–K Reward-to-effort valuation (REV) test 2. H Nose-poke responses at an operant stimulus triggered 1 s tone DS and delivery of chocolate sucrose pellets on a progressive ratio (PR) schedule (5 trials at PR1, 5 × PR5, 5 × PR9, 5 × PR13, etc.). Statistical analysis was conducted using linear mixed models with fixed effects of group (G) and dose (D) and a random effect of mouse subject. I Number of operant responses. J Number of chocolate pellets earned. K Final ratio attained. L–P REV test 3 with freely-available normal food pellet. L A pellet of normal food provided a low-reward/low-effort choice to operant responding for chocolate pellets. M Number of operant responses. N Number of chocolate pellets earned. O Final ratio attained. P Weight of normal pellet eaten.

Fig. 3. Effects of chronic social stress and GPR52 inverse agonist on reward effortful motivation and nucleus accumbens dopamine release.

A Experimental design. Surgery + R + AAVV: stereotactic surgery, recovery, expression of AAV vector; C + OF: conditioning sessions with patch cord attached to optic fibre; SIG: fibre photometry signal test; REV: reward-to-effort valuation test; VEH + REV/CPD + REV: each mouse given 1 REV test with VEH and 1 REV test with GPR52-IA using a counterbalanced cross-over design. For other abbreviations, see Fig. 1. B CSS procedure. C Reward-to-effort valuation (REV) test with fibre photometry. Behavior: Statistical analysis was conducted using 2-way ANOVA with a between-subject factor of group (G) and a within-subject factor of GPR52-IA dose (D). Significant interaction effects were analyzed using Sidak’s test. D Number of operant responses. E Number of gustatory rewards earned. F Final ratio attained. G Operant phase duration i.e., time from 1st until 5th operant response. H Discriminative stimulus (DS) phase duration i.e., time from DS onset until feeder response. NAc DA activity: Statistical analysis was conducted using linear mixed models with fixed effects of group (G), dose (D) and interval or time (T, S) and a random effect of mouse subject. Significant main or interaction effects were analyzed using Sidak’s test. *p < 0.05, ***p < 0.001. I Schematic showing unilateral injection site of AAV GRAB-DA Sensor in NAc, and fibre optic probe implantation directly dorsal to the injection site. J PR 5 operant phase z-scored DA activity following time-normalization using 10 equal intervals. For each mouse, the mean score for PR 5 trials was calculated and data are given as mean ± s.e.m. per group. K PR 5 DS phase z-scored DA activity following time-normalizaion using 10 equal intervals. For each mouse, the mean z-score for PR 5 trials was calculated and data are given as mean ± s.e.m. per group, as well as the overall mean and individual scores per group. L PR 5 feeder phase z-scored DA activity divided into 10 intervals of 0.5 s. For each mouse, the mean score for PR 5 trials was calculated and data are given as mean ± s.e.m per group, as well as the overall mean and individual scores per group. M DS-phase inter-individual relationship between absolute change (GPR52-IA test – VEH test) in NAc DA activity scores and rewards earned in CON mice. N DS-phase inter-individual relationship between absolute change (GPR52-IA test – VEH test) in NAc DA activity scores and rewards earned in CSS mice.

Fig. 4. Circuit diagrams for proposed effects of chronic social stress and GPR52-inverse agonism on dopamine signaling in nucleus accumbens.

The current findings are interpreted within the framework of the indirect NAc pathway of VTA regulation. It should be noted that the depiction that VP GABA neurons that are post-synaptic to NAc D2R MSNs themselves project to VTA or RMTg is an assumption, albeit widely accepted as an integral component of the indirect pathway. A Basal state: an adaptive level of firing by VTA DA neurons results in adaptive DA release on to NAc MSNs. NAc D2R-MSNs binding of DA reduces their firing and inhibition of VP GABA neurons. VP GABA neurons exert adaptive inhibition of VTA GABA interneurons and RMTg GABA neurons. VTA/RMTg GABA (inter)neurons exert adaptive inhibition on VTA DA neurons. Appetitive behavior leading to an incentive stimulus is processed as primarily rewarding by BA glutamate aversion neurons (A) and reward neurons (R) projecting to NAc D2R- and D1R-MSNs, respectively: phasic DA release is adaptive and incentive motivation is high. B Chronic stress state: a low level of firing by VTA DA neurons results in low DA release on to NAc MSNs. NAc D2R-MSNs binding low DA increases their firing and inhibition of VP GABA neurons. VP GABA neurons exert low inhibition of VTA GABA interneurons and RMTg GABA neurons. VTA/RMTg GABA (inter)neurons exert high inhibition of VTA DA neurons. Appetitive behavior leading to an incentive stimulus is processed as primarily aversive by BA A and R projecting to NAc D2R- and D1R-MSNs, respectively: phasic DA release is low and incentive motivation is low. C Chronic stress state and GPR52 inverse agonist: a low level of firing by VTA DA neurons results in reduced DA release. NAc D2R-MSNs binding low DA increase their firing but inhibition of VP GABA neurons is blocked by GPR52 inverse agonist. VP GABA neurons exert adaptive inhibition of VTA GABA interneurons and RMTg GABA neurons. VTA/RMTg GABA (inter)neurons exert low inhibition of VTA DA neurons. An adaptive level of firing by VTA DA neurons results in adaptive DA release on to NAc MSNs. Despite appetitive behavior leading to an incentive stimulus being processed as primarily aversive by BA A and R projecting to NAc D2R- and D1R-MSNs, respectively: phasic DA release is adaptive and incentive motivation is high. BA basal amygdala, R glutamate reward neuron, A glutamate aversion neuron, NAc nucleus accumbens, D1R medium spiny neuron expressing dopamine receptor 1, D2R medium spiny neuron expressing dopamine receptor 2, VP ventral pallidum, VTA ventral tegmental area, RMTg rostro-medial tegmentum.