Deep brain stimulation of the mesencephalic locomotor centre induces stimulation-dependent behavioural states beyond locomotion

Deep brain stimulation (DBS) of the mesencephalic locomotor region (MLR) has been explored to treat gait disturbances. However, clinical outcomes of MLR-DBS have been disappointing overall, with only modest benefits in some individuals and unwanted effects, including anxiety, reported in others. Preclinical studies in several species, by contrast, show that MLR-DBS can improve locomotor and gait deficits, although defensive behaviours in response to stimulation have also been reported in some species. To investigate this discrepancy, we examined frequency- and amplitude-dependent effects of MLR-DBS in healthy rats, combining semi-supervised and unsupervised behavioural analyses. High-frequency, high-amplitude DBS (80-130 Hz), but not low-frequency stimulation (20-60 Hz), elicited hyperlocomotion intermixed with acute defensive-like behaviours. Low-frequency stimulation instead promoted phases of immobility. Consistently, only high-frequency DBS induced region-specific c-Fos expression in the MLR. Complex behaviours, including hyperlocomotion, rearing, tail rattling, and periods of immobility, were most pronounced in animals with the DBS electrode tip localized to the cuneiform nucleus (CnF) of the MLR. Using AAV tracer constructs for bright labelling of CaMKII-positive neurons and their axons, we identified prominent ascending projections from the CnF to the thalamus, substantia nigra pars compacta, zona incerta, hypothalamus, subthalamic nucleus, and central amygdala (CeA). Retrograde tracing confirmed the CnF-to-CeA projection independently. These findings show that the MLR is anatomically connected to higher-order centres linking motor and defensive networks, with DBS frequency and amplitude critically shaping behavioural outcomes that extend beyond locomotion.