DISTINCT CONNECTIVITY AND FUNCTIONAL CHARACTERIZATION OF HYBRID D1/D2-SPNS IN THE DORSAL STRIATUM

The striatum is the main input nucleus of the Basal Ganglia (BG), critical for motor control and reward-based learning, and is linked to various neuropsychiatric disorders. Striatal projection neurons (SPNs) are classically categorized into two pathways based on their projection targets and dopamine receptor expression: D1- (direct) and D2- (indirect) SPNs. Our previous work identified a third hybrid subpopulation co-expressing both receptors (D1/D2-SPNs), which constitutes an additional indirect pathway and is necessary to modulate the pro- and antikinetic functions of D1- and D2-SPNs, respectively. However, this population remains poorly characterized. Here, we aim to map the connectivity of D1/D2-SPNs within the BG network and identify their upstream inputs using modified monosynaptic rabies retrograde tracing in the dorsal striatum (DS). Furthermore, we investigate their functional role during naturalistic behaviors using in vivo Ca²⁺ imaging to record D2^only- and hybrid D1/D2-SPN activity simultaneously. Our retrograde tracing results reveal that, unlike canonical SPNs, D1/D2-SPNs receive preferential monosynaptic inputs from the striatum itself, followed by the cortex and the thalamus. Furthermore, functional imaging during the Open Field Test shows that the activity of these hybrid neurons is not modulated by locomotion or speed. In contrast, our results suggest a specific role in anxiety-related behaviors, as evidenced by activity patterns observed during the Elevated Plus Maze and Fear Conditioning tasks. These results advance our understanding of striatal circuitry and how SPN subpopulations coordinate during behavior, with potential implications for neuropsychiatric research.