Dopamine serves as an independent gain regulator to shape striatal control of movement

Dopamine (DA) drives locomotion by modulating striatal activity in the dorsolateral striatum (DLS). How DA regulates motor function on subsecond timescales remains poorly understood. To address this question, we performed spectrally resolved triple-color fiber photometry, allowing us to simultaneously monitor DA, glutamatergic inputs, and pathway-specific spiny projection neuron (SPN) activity in freely moving mice during spontaneous behaviors as measured by unsupervised behavior quantifications. We showed that DA dynamics were temporally distinct from spontaneous locomotor kinematics or behavioral states. Instead, peri-event DA levels predicted SPN input-output (I/O) efficacy, defined as SPN activity relative to glutamatergic input, with opposite relationships across pathways: higher DA predicted enhanced efficacy in direct-pathway SPNs but reduced efficacy in indirect-pathway SPNs. Paired with unsupervised behavior quantification, elevating extracellular DA with methylphenidate, a DA re-uptake inhibitor, shifted direct-pathway SPNs toward higher I/O gain and indirect-pathway SPNs toward lower neuronal activity outputs, while biasing behavior toward selected mobile and turning states. Reserpine administration, which mediates DA vesicular depletion, resulted in the opposite efficacy shifts and increased occupancy of immobile states. Together, these findings support that DA does not simply encode spontaneous movement, but acts as a pathway-specific gain controller that dynamically tunes the transformation of glutamatergic input into SPN output to bias locomotor-state transitions in vivo .