Modeling long-range human nigrostriatal connectivity in vitro reveals striatal target-dependent maturation of dopamine neurons

Abstract The human nigrostriatal pathway, comprising dopaminergic neurons in the ventral midbrain that project to the dorsolateral striatum, is essential for motor control and selectively vulnerable in Parkinson’s disease. How this circuit assembles during human development remains poorly understood, and in vitro models capturing its long-range connectivity and spatial organization have been lacking. Here, we establish a compartmentalized, human stem cell-based model of the nigrostriatal pathway by integrating ventral midbrain and striatal organoids within a custom-engineered microfluidic device that confines cell bodies while guiding axonal growth to recapitulate key aspects of in vivo topography. Functional connectivity was confirmed by retrograde rabies tracing, and optogenetic and pharmacological stimulation. Single-nucleus transcriptomic analysis reveals that axonal target engagement with spatially separated striatal neurons promotes maturation of human dopaminergic neurons and activates transcriptional programs associated with neurotransmitter secretion. Our study establishes a biologically relevant in vitro system for investigating how long-range connectivity regulates neuronal assembly in the nigrostriatal circuit and for modelling key pathological features of Parkinson’s disease.