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

Abstract

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.

Keywords: Parkinson disease; deep brain stimulation; optogenetics; striatum; vesicular transporter.

Fig. 1.

The Pitx2-Cre transgene is expressed in the STN and mediates a shift in Vglut2 mRNA levels and distribution. (A) Immunohistochemistry for β-gal (nuclear, Left) and GFP (projections, Right) on coronal mouse Pitx2-Cre^Tau-mGFP sections shows Pitx2-Cre–expressing cell nuclei in the STN (Left) and their projections to the EP (Right) target area. (B) Representative examples of in situ hybridization for Vglut2 (magenta) and Pitx2 (green) mRNA in STN (arrows) of Ctrl (Upper row) and cKO (Lower row), with a merge to the left. (C) Superposition of monochrome in situ images of STN (Pitx2 mRNA, Top row; Vglut2, Middle row) with correlation values indicated according to correlation color scale to the right. mRNA level correlation analysis (Pitx2 mRNA on x axis; Vglut2 on y axis) from representative examples of Ctrl (Left) and cKO (Right). Pearson correlation coefficients for Ctrl and cKO r(Ctrl) = 0.8645 and r(cKO) = 0.71401, respectively. (D) Histograms of quantitative distribution of mRNA intensity; average expression level marked by yellow line, expression detection limit marked by gray dotted line. cKO, conditional knockout; Ctrl, control.

Fig. 2.

Attenuation of Vglut2 expression in the STN severely impairs synaptic communication postsynaptically. (A) (Left) Diagram showing stimulation and recording electrode placement in parasagittal slices containing the STN, EP, and SNr. (Right) Representative examples of synaptic currents elicited by STN stimulation (10% above threshold) in SNr and EP cells in control and cKO mice. (B) Mean EPSC amplitude, current density, and stimulation threshold for EP and SNr cells in control (black bars) and cKO (red bars) mice. (C) Photomicrograph showing representative Pitx2-Cre–driven ChR2 expression (through its reporting protein YFP) selectively in the STN with high magnification in inset. Dentate gyrus (DG) and thalamus (Th) are indicated for reference. (D) Example of extracellular recordings in the STN of control and cKO mice before and during light stimulation. (E) Single units isolated from EP recordings before and during light stimulation. (F) Summary of firing frequency in STN and EP recordings before and during light stimulation in control (black bars) and cKO (red bars) mice. *P < 0.05; **P < 0.001. cKO, conditional knockout; Ctrl, control.

Fig. 3.

Normal motor coordination and gait, and increased locomotion. (A) Fine motor coordination assessment on balance beam as measured by the time (seconds) needed to cross the beam. (B) Crude motor coordination assessment on rotarod, presented as mean of the rounds per minute (rpm) reached in three trials per day per animal on three consecutive days. (C) Illustration of the mouse stride cycle. (D) Stride cycle analysis on automated treadmill using averaged values for all four limbs for statistical analysis. Each mouse was trained to walk on the treadmill at speeds 10 and 15 cm/s and subsequently tested at 25 cm/s. (E) Horizontal (locomotion) and vertical (rearing) baseline activity, as well as resting (corner activity) during 60-min recordings in an open field setting, represented as number of photobeam interruptions. (F) Locomotion over 120-min recordings before (white bars) and after (gray bars) saline (Left) and amphetamine (Right) injections; 30 min preinjection, 90 min postinjection. *P < 0.05. cKO, conditional knockout; Ctrl, control.

Fig. 4.

Altered DAT levels and reduced dopamine clearance. (A) Illustration of areas selected for autoradiographic analysis: dorsomedial (DM), dorsolateral (DL), ventromedial (VM), and ventrolateral (VL) striatal area and the substantia nigra pars compacta (SNc). (B) Specific binding capacity levels expressed as percent of control for DAT-specific [¹²⁵I]RTI-121 ligand; for corresponding quantification of dopamine receptor 1 and 2 (D1R and D2) binding, see Fig. S5. (C) High-speed in vivo chronoamperometry recordings in the dorsal striatum of anesthetized mice. (D) KCl-evoked endogenous dopamine release and subsequent clearance: amplitude, peak area, clearance rate, and T₈₀. Note that T₈₀ is significantly longer in cKO mice. (E) DAT-mediated clearance upon exogenous application of dopamine (maximum amplitude 2 μM) into the striatum; representative sample peaks of control (white) and cKO (gray). Note that dopamine clearance [represented by the time to reabsorb 80% of exogenous dopamine (T₈₀)] is significantly prolonged in cKO mice. (F). Group averages for clearance rate (micromoles per second) (Left) and T₈₀ (seconds) (Right). *P < 0.05; **P < 0.01. cKO, conditional knockout; Ctrl, control.