A VTA-pontine GABA pathway biases backward locomotion via local and distal inhibition

Locomotor direction in mammals is implemented by descending circuits, yet how midbrain selection systems bias directional motor output remains unclear. Here we functionally define a projection-defined inhibitory pathway from ventral tegmental area to the oral pontine reticular nucleus (VTAPnO) whose activation is sufficient to drive backward locomotion. Using single-neuron juxtacellular labeling, circuit-targeted optogenetics, ex vivo electrophysiology, and in vivo recordings, we show that a subpopulation of TH- (non-dopaminergic) VTA neurons projects to PnO while retaining local intra-VTA collaterals that form monosynaptic GABAA synapses. Somatic activation reliably induced backward locomotion, and selective stimulation of VTAPnO terminals reproduced the effect, supporting convergent local and projection-mediated engagement. Pathway recruitment produced a rapid, transient increase in dopaminergic single-unit activity in acute recordings and elicited frequency-dependent increases in dopaminergic population calcium signals measured by fiber photometry in awake mice. During forced locomotion, chronically recorded VTAPnO neurons were preferentially engaged during reverse compared to forward rotations and displayed direction-tuned temporal profiles. Together, these findings reveal a dual local-projection inhibitory architecture through which a projection-defined midbrain pathway can bias locomotor direction through a defined premotor node.