Muscarinic and NMDA Receptors in the Substantia Nigra Play a Role in Reward-Related Learning

Abstract

Background: Reward-related learning, where animals form associations between rewards and stimuli (i.e., conditioned stimuli [CS]) that predict or accompany those rewards, is an essential adaptive function for survival.

Methods: In this study, we investigated the mechanisms underlying the acquisition and performance of conditioned approach learning with a focus on the role of muscarinic acetylcholine (mACh) and NMDA glutamate receptors in the substantia nigra (SN), a brain region implicated in reward and motor processes.

Results: Using RNAscope in situ hybridization assays, we found that dopamine neurons of the SN express muscarinic (mACh5), NMDA2a, NMDA2b, and NMDA2d receptor mRNA but not mACh4. NMDA, but not mACh5, receptor mRNA was also found on SN GABA neurons. In a conditioned approach paradigm, rats were exposed to 3 or 7 conditioning sessions during which light/tone (CS) presentations were paired with delivery of food pellets, followed by a test session with CS-only presentations. Intra-SN microinjections of scopolamine (a mACh receptor antagonist) or AP-5 (a NMDA receptor antagonist) were made either prior to each conditioning session (to test their effects on acquisition) or prior to the CS-only test (to test their effects on expression of the learned response). Scopolamine and AP-5 produced dose-dependent significant reductions in the acquisition, but not performance, of conditioned approach.

Conclusions: These results suggest that SN mACh and NMDA receptors are key players in the acquisition, but not the expression, of reward-related learning. Importantly, these findings redefine the role of the SN, which has traditionally been known for its involvement in motor processes, and suggest that the SN possesses attributes consistent with a function as a hub of integration of primary reward and CS signals.

Keywords: Conditioned reinforcement; NMDA; mACh receptor; reward-related learning; substantia nigra.

Figure 1.

A Representative image of the rat midbrain under 4× magnification illustrating the substantia nigra pars compacta (SNc) and location where images were taken from. (B–D) Representative images under 40× magnification illustrating that Dopamine transporter, DAT(Slc6a3)-positive neurons in the SNc coexpress: (B) NMDA2a (Grin2a) and mACh5 (Chrm5) mRNA, (C) NMDA2b (Grin2b) and mACh5 (Chrm5), and (D) NMDA2d (Grin2d) mRNA.

Figure 2.

(A) Representative image of the rat midbrain under 4× magnification illustrating the substantia nigra pars reticulata (SNr) and location where images were taken from. (B–D) Representative images under 40× magnification illustrating that glutamic acid decarboxylase 67, GAD67 (GAD1)-positive neurons in the SNr express: (B) NMDA2a (Grin2a), (C) NMDA2b (Grin2b), (D) NMDA2d (Grin2d) mRNA but not mACh5 (Chrm5) mRNA.

Figure 3.

(A) Representative image of the rat midbrain under 4× magnification illustrating the substantia nigra pars compacta (SNc) and location where images were taken from. (B–D) Representative images under 40× magnification illustrating that DAT(Slc6a3)-positive neurons in the SNc express: (B) NMDA2a (Grin2a), (C) NMDA2b (Grin2b), (D) NMDA2d (Grin2d) mRNA but not mACh4 (Chrm4) mRNA.

Figure 4.

(A) Representative image of the rat midbrain under 4× magnification illustrating the substantia nigra pars reticulata (SNr) and location where images were taken from. (B–D) Representative images under 40× magnification illustrating that GAD67 (GAD1)-positive neurons in the SNr express: (B) NMDA2a (Grin2a), (C) NMDA2b (Grin2b), and (D) NMDA2d (Grin2d) mRNA but not mACh4 (Chrm4) mRNA.

Figure 5.

A. Representative images under 40× magnification illustrating that nucleus accumbens (NAc) neurons express mACh4 (Chrm4) and NMDA2b (Grin2b) mRNA.

Figure 6.

(A) Schematic timeline of behavioral experimentation testing the effects of intra-substantia nigra (SN) scopolamine or AP-5 on the acquisition of conditioned approach. (B) Intra-SNr scopolamine during conditioning caused significant reductions in CS-preCS difference scores during the CS-only test (C) without affecting overall non-CS nose poke responding. (D) Intra-SNr AP-5 during conditioning caused significant reductions in CS-preCS difference scores during the CS-only test (E) without affecting overall non-CS nose poke responding. (F) Post-experimental histology illustrating cannula placements in the rat SNr. *P < .05 compared with 0 µg/0.5µL/side dose; & session main effect at P < .05 indicates the timing of injection.

Figure 7.

(A) Schematic timeline of behavioral experimentation testing the effects of intra-SN scopolamine or AP-5 on the expression of conditioned approach. (B) Intra-SNr scopolamine during CS-only test had no effect on CS-preCS difference scores (C) even though it increased non-CS nose poke responding. (D) Intra-SNr AP-5 during the CS-only test had no effect on CS-preCS difference scores (E) even though it significantly increased non-CS nose poke responding. (F) Post-experimental histology illustrating cannula placements in the rat SNr. (G) Representative image illustrating cannula placement. *P < .05 as compared to 0 µg/0.5µL/side dose; & session main effect at P < .05 indicates the timing of injection.

Figure 8.

Our circuit model of reward-related learning. We propose that dopamine (DA) neurons of the substantia nigra pars compacta (SNc) coexpress muscarinic acetylcholine (mACh) and NMDA receptors. Convergent cholinergic and glutamatergic neurotransmission via mACh and NMDA glutamate receptors in the SN is needed for the acquisition of conditioned approach to occur.