The contribution of NMDA receptor signaling in the corticobasal ganglia reward network to appetitive Pavlovian learning

Abstract

NMDA receptors (NMDARs) contribute to phasic transmission and synaptic plasticity and are thought to be important for learning. To better understand where NMDAR signaling is necessary for learning, we combined viral genetic strategies with genetic mouse models to investigate the contribution of NMDARs in the dopamine system to appetitive Pavlovian conditioning. NMDAR signaling in dopamine neurons was not required for Pavlovian conditioning; however, NMDARs in D(1) dopamine receptor (D(1)R)-expressing medium spiny neurons (MSNs), which receive input from dopamine neurons, were critical for this type of learning. NMDAR signaling was also required in brain regions that project to dopamine neurons, because removing NMDARs from afferent neurons to the ventral tegmental area (VTA) also prevented learning. This effect was likely attributable to loss of NMDAR signaling in the neurons of the prefrontal cortex (PFC), because learning could be restored in these animals by rescuing NMDAR expression in the PFC. Moreover, removing NMDARs exclusively from the PFC also prevented learning. Our findings suggest that NMDARs in neurons that project to and receive projections from the VTA are necessary for Pavlovian conditioning and specifically implicate the PFC and D(1)R-expressing MSNs in associative learning.

Figure 1.

Mice lacking NMDARs in dopamine neurons learn normally during Pavlovian conditioning. A, Gray dots in the midbrain indicate that only the dopamine neurons in this brain region lack NMDARs in DAT–Cre; NR1–KO mice. B, Control mice (n = 11) increase their CS–HE rate relative to their baseline ITI–HE rate during training (CS vs ITI; F_(4,80) = 6.2, ***p < 0.001). C, DAT–Cre; NR1–KO mice (n = 11) also increase their CS–HE rate relative to ITI–HE rate (CS vs ITI; F_(4,64) = 6.2, ***p < 0.001). D, Both groups increase their CA during learning.

Figure 2.

Pavlovian conditioning is disrupted in mice lacking NMDARs in striatal MSNs and D₁R-expressing neurons. A, Large gray dots indicate the brain region lacking NMDARs (striatal MSNs) in GPR88–Cre; NR1–KO mice. B, Control mice (n = 9) increased their CS–HE rate relative to their ITI–HE rate (CS vs ITI; F_(4,64) = 12.6, ***p < 0.001). C, CS–HE and ITI–HE rates did not differ during training in GPR88–Cre; NR1–KO mice (n = 9). D, CA score increased in control but not GPR88–Cre; NR1–KO mice during training (KO vs control; F_(4,64) = 5.4, ***p < 0.001). E, Small gray dots indicate that D₁R-expressing neurons lack NMDARs in D1–Cre; NR1–KO mice. F, Control mice (n = 11) increased their CS–HE rate relative to ITI–HE rate during training (CS vs ITI; F_(4,80) = 6.5, ***p < 0.001). G, CS–HE and ITI–HE rates did not differ during training in D1–Cre; NR1–KO mice (n = 9). H, CA score was significantly attenuated in D1–Cre; NR1–KO mice during training (KO vs control; F_(4,72) = 3.4, *p < 0.05).

Figure 3.

Mice lacking NMDARs in the VTA as well as in VTA-projecting brain regions failed to learn during Pavlovian conditioning. A, Viral injection strategy for CAV–VTA; NR1–KO mice. Solid green shading indicates that most cells within the VTA lack NMDARs, and partially shaded green indicates that only the VTA-projecting neurons lack NMDARs in brain regions outside of the VTA. B, C, CAV–Cre injection is selective for the VTA and leads to viral transduction and activated Cre expression (green) in both TH-positive (red) and TH-negative neurons. Scale bars: B, 500 μm; C, 100 μm. D, CAV–Cre injection into the VTA transduces and activates Cre expression (green) in VTA-projecting PFC neurons. Scale bars: Top, 1 mm; bottom, 200 μm. Blue, DAPI stain. E, Control mice (n = 8) increase their CS–HE rate relative to ITI–HE rate during training (CS vs ITI; F_(4,56) = 5.9, **p < 0.01). F, CAV–VTA; NR1–KO mice (n = 6) fail to increase their CS–HE rate relative to ITI–HE rate. G, Control but not CAV–VTA; NR1–KO mice increase their CA score during training (control vs KO; F_(4,48) = 5.9, ***p < 0.001).

Figure 4.

Mice lacking NMDARs in the PFC do not learn during Pavlovian conditioning. A, Viral injection strategy for generating AAV–PFC; NR1–KO mice. Solid green shading indicates that most cells within PFC injection site lack NMDARs. B, C, AAV–Cre injection into the PFC transduces and activates Cre-mediated recombination in his brain region. Scale bars: B, 1 mm; C, 200 μm. D, Control mice (n = 8) increase their CS–HE rate relative to ITI–HE rate during training (CS vs ITI; F_(4,56) = 2.9, *p < 0.05). E, AAV–PFC; NR1–KO mice (n = 6) fail to increase their CS–HE rate relative to ITI–HE rate. F, Control but not AAV–Cre; NR1–KO mice increase their CA score during training (control vs KO; F_(4,48) = 2.7, *p < 0.05).

Figure 5.

Selectively restoring NMDARs to the VTA-projecting neurons of the PFC rescues learning in CAV–VTA; NR1–KO mice. A, Top, Diagram of AAV–fsNR1 construct; bottom, viral injection strategy for generating CAV–VTA; NR1–PFC–Rescue mice. Solid green shading indicates that most cells within the VTA lack NMDARs, and partially shaded green indicates that only the VTA-projecting neurons lack NMDARs in brain regions outside of the VTA. Partially shaded red region indicates the selective restoration of NMDARs to VTA-projecting PFC neurons. B, CAV–Cre injection into the VTA transduces and activates Cre expression (green) in VTA-projecting PFC neurons. Scale bar, 200 μm. C, AAV–fsNR1 injection into the PFC activates HA–NR1 expression in the PFC (red). D, Overlay of Cre-activated YFP expression (green) and HA–NR1 staining (red) indicates that NR1 is re-expressed in VTA-projecting PFC neurons. E, Control mice (n = 8) increase their CS–HE rate relative to ITI–HE rate during training (CS vs ITI; F_(4,56) = 3.8, **p < 0.01). F, CAV–VTA; NR1–PFC–Rescue mice (n = 7) also increase their CS–HE rate relative to ITI–HE rate during training (CS vs ITI; F_(4,48) = 2.7, *p < 0.05). G, Both groups increase their CA score during learning.