| Abstract: | In Parkinson’s disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.In Parkinson’s disease (PD), midbrain dopaminergic (DA) cells are lost, resulting in motor dysfunction, including severe locomotor deficits (e.g., gait freezing, akinesia, and falls) (1). Growing evidence indicates that part of these deficits can be attributed to changes in the mesencephalic locomotor region (MLR) (refs. 2–7; for review, see ref. 8). This brainstem region plays a key role in locomotor control by sending projections to reticulospinal neurons that carry the locomotor drive to the spinal cord in vertebrates (lamprey: refs. 9 and 10; salamander: refs. 11 and 12; and mouse: refs. 13 to 19; for review, see ref. 20). The DA neurons of the substantia nigra pars compacta (SNc) indirectly control MLR activity through the basal ganglia (15, 21, 22). In parallel, the MLR receives direct DA projections from the SNc (23–26) and from the zona incerta (27). The DA innervation of the MLR degenerates in a monkey model of PD (28). Therefore, the loss of DA cells in PD has major effects on MLR activity. In PD, locomotor deficits are associated with MLR cell loss, abnormal neural activity, altered connectivity, and metabolic deficits, likely resulting in a loss of amplification of the locomotor commands (for review, see ref. 8). Accordingly, motor arrests and gait freezing are associated with a decrease in MLR activity in PD (ref. 29; for review, see ref. 8).One approach to improve locomotor function in PD would be to increase MLR activity. L-DOPA, the gold standard drug used to improve motor symptoms in PD, increases MLR activity and this likely contributes to the locomotor benefits (30). The MLR has been proposed to contribute to the locomotor benefits of deep brain stimulation (DBS) of the subthalamic nucleus (31–33), which has direct and indirect projections to the MLR (refs. 34, 35; for review, see ref. 20). However, the benefits of L-DOPA and subthalamic DBS on locomotor deficits may wane over time, highlighting the need to find new therapeutic approaches (36, 37). Since 2005, the MLR has been explored as a DBS target (38). The results vary, from modest to promising (39, 40). However, the best target in the MLR in PD conditions is not yet identified. The MLR is a heterogeneous structure, with the cuneiform nucleus (CnF) controlling the largest range of locomotor speeds, and the pedunculopontine nucleus (PPN) controlling slow speeds, posture and in some cases locomotor arrests (refs. 15, 17–19; for review, see ref. 20). Human DBS protocols targeted the PPN, but left the CnF unexplored, despite its major importance in locomotor control in animal research (19, 41, 42). In humans, a recent anatomical analysis of DBS electrode position relative to the pontomesencephalic junction suggests that some of the beneficial effects attributed to the PPN could be due to CnF activation (43).To add further complexity, three main cell types are present in the MLR: glutamatergic, GABAergic, and cholinergic cells. It is still unknown which cell type is the best target to improve locomotor function in PD conditions. Optogenetic studies uncovered that glutamatergic cells in the CnF play a key role in generating the locomotor drive for a wide range of speeds (15, 17–19). Glutamatergic cells of the PPN control slower speeds (17, 18) and, in some cases, evoke locomotor arrests (18, 44, 45). The GABAergic cells in the CnF and PPN stop locomotion likely by inhibiting glutamatergic cells (15, 17). The role of the PPN cholinergic cells is not resolved, as their activation can increase or decrease locomotion (refs. 15, 17, 18; for review, see ref. 20). Clinically, DBS likely stimulates all cells around the electrode, including the GABAergic cells that stop locomotion, and this could contribute to the variability of outcomes.Here, we aimed at identifying a relevant target in the MLR to improve the locomotor function in parkinsonian conditions. We hypothesized that the selective activation of CnF glutamatergic neurons should improve locomotor function in a mouse model of PD. We induced parkinsonian conditions in mice by bilaterally injecting into the striatum the neurotoxin 6-hydroxydopamine (6-OHDA), which is well known to damage the nigrostriatal DA pathway and to induce a dramatic decrease in locomotor activity (e.g., refs. 22 and 46). Using in vivo optogenetics in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice), we show that the photostimulation of glutamatergic neurons in the CnF robustly initiated locomotion, reduced immobility, increased the time spent in locomotion, and precisely controlled locomotor speed. Our results should help in defining therapeutic strategies aimed at specifically activating CnF glutamatergic neurons to improve locomotor function in PD using optimized DBS protocols, pharmacotherapy, or future optogenetic tools for human use. |
