MHC class I in dopaminergic neurons suppresses relapse to reward seeking

Abstract

Major histocompatibility complex class I (MHCI) is an important immune protein that is expressed in various brain regions, with its deficiency leading to extensive synaptic transmission that results in learning and memory deficits. Although MHCI is highly expressed in dopaminergic neurons, its role in these neurons has not been examined. We show that MHCI expressed in dopaminergic neurons plays a key role in suppressing reward-seeking behavior. In wild-type mice, cocaine self-administration caused persistent reduction of MHCI specifically in dopaminergic neurons, which was accompanied by enhanced glutamatergic synaptic transmission and relapse to cocaine seeking. Functional MHCI knockout promoted this addictive phenotype for cocaine and a natural reward, namely, sucrose. In contrast, wild-type mice overexpressing a major MHCI gene (H2D) in dopaminergic neurons showed suppressed cocaine seeking. These results show that persistent cocaine-induced reduction of MHCI in dopaminergic neurons is necessary for relapse to cocaine seeking.

Fig. 1. Cocaine- and sucrose-seeking behavior in WT and functional MHCI knockout (KO) mice.

(A) Computed tomography image showing a mouse with a microdialysis probe implanted into the supracerebellar cistern. Arrows indicate perfusion tubing. (B) Cocaine self-administration procedure. (C) Active nose-pokes in WT and β2M^−/−TAP1^−/− mice that self-administered vehicle (Veh) or cocaine (Coc) during the reinstatement period [two-way analysis of variance (ANOVA); genotype: F_(1,77) = 4.2, P < 0.05; treatment: F_(1,77) = 17.2, P < 0.001; genotype × treatment: F_(1,77) = 5.9, P < 0.05; n = 21 for WT-Veh, n = 19 for WT-Coc, n = 21 for KO-Veh, and n = 20 for KO-Coc). (D) Active nose-pokes of WT and β2M^−/−TAP1^−/− mice that self-administered Veh or sucrose (Suc) during the reinstatement period [two-way ANOVA; treatment: F_(1,27) = 5.5, P < 0.05; genotype × treatment: F_(1,27) = 4.5, P < 0.05; n = 8 for WT-Veh, n = 8 for WT-Suc, n = 7 for KO-Veh, and n = 8 for KO-Suc]. All data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2. Cocaine-induced decrease of MHCI expression in the VTA.

(A) Experimenter-administered cocaine injection procedure. (B) Locomotor activity in WT mice injected with Veh or Coc. (C) H2D mRNA levels in the hippocampus (Hip), NAc, VTA, prefrontal cortex (PFC), and amygdala (Amy) of WT mice injected with Veh or Coc (Student’s t test, P < 0.05; n = 8 for Veh and n = 8 for Coc). (D) H2D protein in the VTA of WT mice injected with Veh or Coc (Student’s t test, P < 0.05; n = 8 for Veh and n = 6 for Coc). All data are represented as means ± SEM and labeled as in (B). *P < 0.05.

Fig. 3. Immunohistochemical analysis of MHCI localization in the VTA.

(A) Immunohistochemistry with an antibody against H2D to detect cell surface–expressed protein in the VTA of WT and β2M^−/−TAP1^−/− mice. SNR, substantia nigra reticulata. (B) Western blot analysis of H2D protein in microsomal fractions prepared from the whole brain of WT and β2M^−/−TAP1^−/− mice. (C) Double staining of H2D and TH, a marker of dopaminergic neurons, in the VTA of WT mice. Arrows indicate colocalization between H2D and TH. (D) Double staining of MHCI/H2D with postsynaptic (PSD-95) and presynaptic (synaptophysin) markers in the VTA of WT mice. Arrows indicate colocalization between MHCI and PSD-95. (E) Pearson’s correlation coefficient to quantify the degree of colocalization between MHCI and PSD-95 or synaptophysin. Costes’ test showed statistically significant correlation between MHCI and PSD-95. Data are represented as means ± SEM. Student’s t test, **P < 0.01.

Fig. 4. Ex vivo whole-cell patch-clamp recordings and spine analysis in VTA dopaminergic neurons after an extinction period of cocaine self-administration.

(A) AMPAR/NMDAR ratio in VTA dopaminergic neurons of WT and β2M^−/−TAP1^−/− mice after an extinction period of Veh or Coc self-administration [two-way ANOVA; genotype: F_(1,42) = 5.3, P < 0.05; treatment: F_(1,42) = 10.8, P < 0.01; n = 13 slices for WT-Veh, n = 14 slices for WT-Coc, n = 9 slices for KO-Veh, and n = 10 slices for KO-Coc; VTA slices were prepared from six mice in each group]. (B to D) Representative traces (B), amplitude (C), and frequency (D) of mEPSCs in VTA dopaminergic neurons of WT and β2M^−/−TAP1^−/− mice after an extinction period of Veh or Coc self-administration [two-way ANOVA; amplitude: treatment: F_(1,29) = 10.8, P < 0.001; genotype × treatment: F_(1,29) = 5.1, P < 0.05; frequency: genotype: F_(1,29) = 4.6, P < 0.05; treatment: F_(1,29) = 6.6, P < 0.05; n = 8 slices for WT-Veh, n = 9 slices for WT-Coc, n = 8 slices for KO-Veh, and n = 8 slices for KO-Coc; VTA slices were prepared from four mice in each group]. (E to G) Three-dimensional images of dendritic spines (E) and summary of spine density (F) and head diameter (G) in VTA dopaminergic neurons of WT and β2M^−/−TAP1^−/− mice after an extinction period of Veh or Coc self-administration [two-way ANOVA: spine density: genotype: F_(1,80) = 5.5, P < 0.05; treatment: F_(1,80) = 17.3, P < 0.001; n = 27 slices for WT-Veh, n = 18 slices for WT-Coc, n = 13 slices for KO-Veh, and n = 26 slices for KO-Coc; diameter: treatment: F_(1,83) = 12.7, P < 0.01; genotype × treatment: F_(1,83) = 3.9, P < 0.05; n = 27 slices for WT-Veh, n = 21 slices for WT-Coc, n = 13 slices for KO-Veh, and n = 26 slices for KO-Coc; VTA slices were prepared from six mice in each group]. All data are represented as means ± SEM and labeled as in (A). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. Cocaine-seeking behavior in WT mice overexpressing H2D in VTA dopaminergic neurons.

(A) Recombinant AAV-2 constructs encoding EGFP and H2D under control of the TH promoter (pTH). ITR, inverted terminal repeat; hGHpA, human growth hormone polyA sequence. (B) Immunostaining of EGFP, H2D, and TH in the VTA of mice expressing EGFP (upper) or H2D (lower). TH expression was identified in >90% of neurons expressing these genes. (C) Protein levels of H2D in the VTA of EGFP- or H2D-expressing mice. (D) mRNA levels of H2D in VTA of EGFP- and H2D-expressing mice (Student’s t test, P < 0.001; n = 7 for EGFP and n = 8 for H2D). (E) Active nose-pokes of EGFP- and H2D-expressing mice during reinstatement of cocaine self-administration (Student’s t test, P < 0.05; n = 8 for EGFP and n = 8 for H2D). All data are represented as means ± SEM and labeled as in (D). *P < 0.05 and ***P < 0.001.