Subtype-specific roles of nigrostriatal dopaminergic neurons in motor and associative learning

Aim: Define the subtype-specific contributions of nigrostriatal dopaminergic neurons (DANs) to motor and non-motor behaviors by comparing Calbindin 1-positive (Calb1⁺) and Aldehyde dehydrogenase 1a1-positive (Aldh1a1⁺) DANs. Methods: Intersectional genetic strategy and chemogenetic inhibition were applied to selectively silence Calb1⁺ or Aldh1a1⁺ DANs in mice. An adeno-associated viral vector (AAV-CreOn-FlpOn-hM4Di-P2A-mCherry) was stereotactically delivered into the substantia nigra pars compacta of double knock-in lines ThFlp; Calb1IRESCre or ThFlp; Aldh1a1CreERT2. Following expression, subtype-specific neuronal inhibition was induced with a designer receptor exclusively activated by designer drugs (DREADD) ligand, and the mice were assessed in assays of voluntary movement, motor skill learning, and early associative learning behavior. Results: Chemogenetic inhibition of either Calb1⁺ or Aldh1a1⁺ DANs produced a marked reduction in voluntary movement and impaired acquisition of motor skills, indicating that both subtypes are necessary for normal motor function and learning. In contrast, only inhibition of Calb1⁺ DANs altered early associative-learning performance, revealing a dissociable, subtype-specific role for Calb1⁺ neurons in reinforcement-related behavior that was not observed with Aldh1a1⁺ neuron inhibition. Conclusion: Both Calb1⁺ and Aldh1a1⁺ nigrostriatal DANs are key regulators of movement and motor learning, with Calb1⁺ neurons additionally modulating reward-based associative learning. These findings highlight the functional heterogeneity of nigrostriatal DAN subtypes and identify potential therapeutic targets for addressing motor and non-motor deficits in Parkinson’s disease.