Balanced NMDA receptor activity in dopamine D1 receptor (D1R)- and D2R-expressing medium spiny neurons is required for amphetamine sensitization

Abstract

Signaling through N-methyl-D-aspartate-type glutamate receptors (NMDARs) is essential for the development of behavioral sensitization to psychostimulants such as amphetamine (AMPH). However, the cell type and brain region in which NMDAR signaling is required for AMPH sensitization remain unresolved. Here we use selective inactivation of Grin1, the gene encoding the essential NR1 subunit of NMDARs, in dopamine neurons or their medium spiny neuron (MSN) targets, to address this issue. We show that NMDAR signaling in dopamine neurons is not required for behavioral sensitization to AMPH. Conversely, removing NMDARs from MSNs that express the dopamine D1 receptor (D1R) significantly attenuated AMPH sensitization, and conditional, virus-mediated restoration of NR1 in D1R neurons in the nucleus accumbens (NAc) of these animals rescued sensitization. Interestingly, sensitization could also be restored by virus-mediated inactivation of NR1 in all remaining neurons in the NAc of animals lacking NMDARs on D1R neurons, or by removing NMDARs from all MSNs. Taken together, these data indicate that unbalanced loss of NMDAR signaling in D1R MSNs alone prevents AMPH sensitization, whereas a balanced loss of NMDARs from both D1R and dopamine D2 receptor-expressing (D2R) MSNs is permissive for sensitization.

Fig. 1.

NMDARs on dopamine neurons are not required for AMPH sensitization. (A) AMPH sensitization paradigm. On all days, animals were injected i.p. after 90-min habituation in locomotion chambers, and locomotion was measured for 90 min after injection. (B) Cumulative 90-min locomotor response to AMPH in DAT:NR1 control (n = 13) and DAT:NR1 knockout (n = 13) mice across days. DW, days of withdrawal. Two-way, repeated-measures ANOVA: genotype effect, F(1, 24) = 0.01, P = 0.90; day effect, F(6, 144) = 14.41, P = 0.01; genotype × day effect, F(6, 144) = 0.79, P = 0.58.

Fig. 2.

Removing NMDARs from D1R-expressing neurons impairs AMPH sensitization. (A) Baseline locomotion in D1R:NR1 control and knockout mice; novelty response (distance traveled in 60 min in a novel environment by D1R:NR1 control mice: 230 ± 80 cm vs. D1R:NR1 knockout mice: 200 ± 60 cm), P = 0.74; light cycle, P = 0.22; dark cycle, P = 0.45 by unpaired t tests. (B) Representative traces of NMDAR and AMPAR EPSCs from TdTomato-expressing neurons in the ventral striatum of D1R:NR1 control and knockout mice. (C) Cumulative 90-min locomotor response to AMPH in D1R:NR1 control (n = 9) and knockout (n = 9) mice across days; two-way, repeated-measures ANOVA: genotype effect, F(1, 16) = 9.51, P = 0.007; day effect, F(6, 96) = 6.80, P < 0.0001; genotype × day effect, F(6, 96) = 3.96, P = 0.0014 (^##P < 0.01); **P < 0.01; ***P < 0.001 compared with AMPH day 1 within genotype. (D) Difference between time spent in AMPH-paired and saline-paired compartments at baseline and after 1 d of AMPH pairing in D1R:NR1 control (n = 9) and knockout (n = 10) mice. Two-way, repeated-measures ANOVA: genotype effect, F(1, 17) = 0.78, P = 0.39; day effect, F(1, 17) = 22.56, P = 0.0002; genotype × day effect, F(1, 17) = 4.76, P = 0.04. *P < 0.05 vs. baseline.

Fig. 3.

Restoring NMDAR signaling to the NAc of D1R:NR1 knockouts rescues AMPH sensitization. (A) Schematic diagram of AAV1-fsNR1 virus construct. (B) Western blot of striatal homogenates from two D1R-Cre (−) and two D1R-Cre (+) mice that had received intra-NAc injections of AAV1-fsNR1, probed with anti-HA antibody showing Cre-dependent expression of full-length viral NR1. (C) Cumulative 90-min locomotor response to AMPH from AAV1-fsNR1–injected D1R:NR1 control (n = 5) and knockout (n = 5) mice across days; two-way, repeated-measures ANOVA: genotype effect, F(1, 8) = 0.1246, P = 0.73; day effect, F(6, 48) = 9.10, P < 0.0001; genotype × day effect, F(6, 48) = 0.25, P = 0.96. **P < 0.01, ***P < 0.001 compared with AMPH day 1 within genotype. (D) Immunohistochemistry with anti-HA antibody showing NAc-specific expression of viral NR1. NAc is outlined in white. A DAPI counterstain was used.

Fig. 4.

Removing NMDAR signaling from both classes of MSNs is permissive for AMPH sensitization. (A) Schematic diagram of AAV1-Cre-GFP virus construct. (B) Cumulative 90-min locomotor response to AMPH from AAV1-Cre-GFP–injected D1R:NR1 control (n = 9) and knockout (n = 5) mice across days; two-way, repeated-measures ANOVA: genotype effect, F(1, 12) = 3.343, P = 0.09; day effect, F(6, 72) = 22.08, P < 0.0001; genotype × day effect, F(6, 72) = 4.23, P = 0.01 (^##P < 0.01). *P < 0.05, ***P < 0.001 compared with AMPH day 1 within genotype. (C) GFP fluorescence showing NAc-specific injection of AAV1-Cre-GFP into D1R:NR1 knockout mice. NAc is outlined in white. A DAPI counterstain was used. (D) Cumulative 90-min locomotor response to AMPH from control mice (n = 14) and mice in which NR1 is inactivated in the NAc (n = 12) across days; two-way, repeated-measures ANOVA: genotype effect, F(1, 23) = 2.00, P = 0.17; day effect, F(6, 138) = 26.59; P < 0.0001; genotype × day effect, F(6, 138) = 1.07, P = 0.39. *P < 0.05, ***P < 0.001 compared with AMPH day 1 within genotype. (E) GFP fluorescence showing NAc-specific injection of AAV1-Cre-GFP into Grin1^lox/lox mice. NAc is outlined in white. A DAPI counterstain was used. (F) Western blot of striatal homogenates from two GPR88:NR1 control and two GPR88:NR1 knockout animals probed with anti-NR1 antibody. (G) Cumulative 90-min locomotor response to AMPH from GPR88:NR1 control (n = 8) and knockout (n = 7) mice across days; two-way, repeated-measures ANOVA: genotype effect, F(1, 13) = 4.163, P = 0.06; day effect, F(6, 78) = 26.96, P < 0.0001; genotype × day effect, F(6, 78) = 1.81, P = 0.11. *P < 0.05, **P < 0.01, ***P < 0.001 compared with AMPH day 1 within genotype.