Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.The subthalamic nucleus (STN) has long been a structure of interest for researchers and clinicians alike. There is ample evidence that high-frequency stimulation of the STN improves symptoms such as tremor, rigidity, and slowness of movement, so called bradykinesia, in patients with Parkinson disease (see ref. 1 for review), but the mechanism through which this is achieved is still unknown. Some studies suggest that electrical stimulation causes a hyperexcitation of this structure (2), whereas others find evidence that the opposite is true (3–5). Other possible interpretations include the activation of the zona incerta, a neighboring white-matter structure (6) or of fibers coming from the motor cortex (7). Bilateral lesions of the STN improve locomotion (8), a result that is consistent with the inactivation hypothesis. However, previous studies have also found cognitive side effects when using high-frequency stimulation of the STN (9), findings supported by lesion studies in experimental animals, which led to abnormalities in operant tasks involving attention and impulsivity (10, 11). The projections of the STN to other regions help explain the multiple roles of this structure: It sends projections to other targets in the basal ganglia, such as the internal segment of the globus pallidus [also termed the entopeduncular nucleus (EP) in rodents] and the substantia nigra pars reticulata (SNr) (12, 13). The STN is also part of a circuit that includes the prefrontal cortex and the nucleus accumbens (14). It is currently unknown, however, whether these different roles reflect a heterogeneous population of cells, characterized by distinct gene expression. If that is the case, it would allow direct control over each cell population, facilitating the investigation of their respective roles. In rodents, the STN is believed to be composed solely of glutamatergic neurons, characterized by expression of the subtype 2 Vesicular glutamate transporter (Vglut2), whereas the other two subtypes (Vglut1 and Vglut3) have not been detected (15, 16). Selective targeted deletion of Vglut2 expression in this nucleus would therefore provide a specific loss-of-function model that would bypass a common problem presented by traditional lesions with pharmacological agents, which have patterns of diffusion that likely affect surrounding structures (17). It is known, however, that Vglut2 is expressed in many other parts of the brain (18), and a complete knockout in the mouse is not viable (19, 20). There is also evidence that the promoter driving expression of the Paired-like homeodomain 2 (Pitx2) gene is strong in the mouse STN (21) but is also not specific to this structure and a full knockout of Pitx2 expression results in premature death (22). To achieve the desired level of specificity, using a conditional knockout technique previously used to eliminate glutamatergic transmission in other cell types (23), we crossed Pitx2-Cre and Vglut2-lox mice, producing Vglut2^{f/f;Pitx2-Cre} conditional knockout (cKO) mice in which Vglut2 expression in the STN was strongly reduced in comparison with expression levels in littermate control mice. To understand the physiological contribution of the selected subpopulation of STN cells, we characterized these cKO mice with regard to anatomical, electrophysiological, and molecular properties, as well as their performance in a range of behavioral tasks.