Optogenetic Stimulation of GABAergic Neurons in the Globus Pallidus Produces Hyperkinesia

Abstract

The globus pallidus (GP) is emerging as a critical locus of basal ganglia control of motor activity, but the exact role of GABAergic GP neurons remain to be defined. By targeted expression of channelrhodopsin 2 (ChR2) in GABAergic neurons using the VGAT-ChR2-EYFP transgenic mice, we showed that optogenetic stimulation of GABAergic neurons in the right GP produced hyperkinesia. Optogenetic stimulation of GABAergic GP neurons increased c-Fos-positive cells in GP, M1 cortex, and caudate-putamen (CPu), and decreased c-Fos-positive cells in entopeduncular nucleus (EPN), compared to the contralateral hemisphere. In agreement with the canonical basal ganglia model. Furthermore, we delivered AAV-CaMKIIα-ChR2-mCherry virus to the excitatory neurons of the subthalamic nucleus (STN) and selectively stimulated glutamatergic afferent fibers from the STN onto the GP. This optogenetic stimulation produced abnormal movements, similar to the behaviors that observed in the VGAT-ChR2-EYFP transgenic mice. Meanwhile, we found that the c-Fos expression pattern in the GP, M1, STN, EPN, and CPu produced by optogenetic activation of glutamatergic afferent fibers from the STN in GP was similar to the c-Fos expression pattern in the VGAT-ChR2-EYFP transgenic mice. Taken together, our results suggest that excess GP GABAergic neurons activity could be the neural substrate of abnormal involuntary movements in hyperkinetic movement disorders. The neural circuitry underlying the abnormal involuntary movements is associated with excessive GP, M1, CPu activity, and reduced EPN activity. Inhibition of GP GABAergic neurons represents new treatment targets for hyperkinetic movement disorder.

Keywords: GABAergic neurons; globus pallidus; hyperkinesia; movement disorders; optogenetic stimulation.

Figure 1

ChR2 activation of the GP GABAergic neurons induced dystonia-like behaviors in VGAT-ChR2-EYFP transgenic mice. (A) Specific expression of ChR2 in GP GABAergic neurons in VGAT-ChR2-EYFP transgenic mice. The confocal images show that GP GABAergic neurons expressed ChR2-EYFP and were co-stained with GAD67. (B) The mouse did not show any abnormal performance before light stimulation (upper panel) and showed torsion of the neck and forelimb after light stimulation of the right GP (lower panel). The symptoms could be seen more clearly in the video (Video S1). (C) The percentage of the 30-s period the animal spent engaging in each behavior. During optical stimulation of the right GP, the VGAT-ChR2-EYFP transgenic mice displayed more grooming, licking, chewing, dystonia-like behaviors (torsion of the neck and left forelimb) and circling left, and displayed less resting, exploration and circling right, compared to that prior to light stimulation [**p < 0.01, ***p < 0.001, n = 8, using a paired, two-tailed t-test; grooming: t₍₇₎ = −4.989, p = 0.002; licking: t₍₇₎ = −10.728, p < 0.001; chewing: t₍₇₎ = −18.466, p < 0.001; torsion spasm: t₍₇₎ = −24.826, p < 0.001; circling left: t₍₇₎ = −10.458, p < 0.001; resting: t₍₇₎ = 5.255, p = 0.001; exploration: t₍₇₎ = 6.844, p < 0.001; circling right; t₍₇₎ = 5.624, p = 0.001]. (D) EEG recording in the M1 cortex from a mouse that developed dystonia-like behaviors. The EEG signal in the M1 cortex did not show any sign of epileptic activity. The red dashed line represents the time of optical stimulation. Power spectrum of EEG signals recorded before, during, and after stimulus are depicted. There was no apparent increased power for the frequency range from 0.5 to 40 Hz during the stimulus.

Figure 2

ChR2 stimulation of the GP GABAergic neurons produced the network level changes of basal ganglia circuit as indicated by c-Fos expression in the GP, M1, EPN, STN, and CPu. (A) We detected c-Fos immunoreactivity in the light-exposed ipsilateral and contralateral hemispheres of VGAT-ChR2-EYFP transgenic mice, as well as in the light-exposed hemisphere of wild-type mice (control). (B) The c-Fos-positive neurons in the GP were all GAD67-positive neurons. (C) A bar graph of c-Fos-positive cell counts in the GP, M1, EPN, STN, and CPu. Asterisks indicate significant difference between the c-Fos-positive cells in ipsilateral hemispheres and contralateral hemispheres to optical stimulation {n = 10, two-way ANOVA followed by post-hoc Tukey's test. There was a significant effect of ChR2 stimulation [F_(2,18) = 20.377, p < 0.001], and brain region effect [F_(4,36) = 54.76, p < 0.001]. A ChR2 stimulation–brain region interaction [F_(8,72) = 28.22, p < 0.001] occurred. Post-hoc comparison: GP (right hemisphere light vs. left hemisphere): ***p < 0.001; M1 (right hemisphere light vs. left hemisphere): ***p < 0.001; EPN (right hemisphere light vs. left hemisphere): ***p < 0.001; CPu (right hemisphere light vs. left hemisphere): ***p < 0.001}.

Figure 3

ChR2 stimulation of glutamatergic afferent fibers from the STN in the GP produced similar abnormal movements. (A) ChR2-mCherry was selectively expressed in the STN but not in other nearby regions. ChR2-mCherry was also expressed in the two other remote brain regions: GP and EPN, but not in the M1. The selected schematics were adapted from the mouse brain in Stereotaxic Coordinates (Paxinos and Franklin, 2001). (B) ChR2-mCherry-expressing STN neurons co-stained with the excitatory neuron-specific marker CaMKIIα. (C) Schematic of the optical stimulation of glutamatergic afferent fibers from the STN in the GP. (D) The percentage of the 30-s period the animal spent engaging in each behavior. During optical stimulation of the right GP, the mice displayed more grooming, dystonia-like behaviors (torsion of the neck and left forelimb) and circling right, and displayed less resting and exploration [**p < 0.01, ***p < 0.001, n = 8, using a paired, two-tailed t-test; grooming: t₍₇₎ = −6.859, p < 0.001; torsion spasm: t₍₇₎ = 15.317, p < 0.001; resting: t₍₇₎ = 8.793, p < 0.001; exploration: t₍₇₎ = 10.604, p < 0.001; circling left: t₍₇₎ = 4.660, p = 0.002; circling right: t₍₇₎ = −10.817, p < 0.001; chewing: t₍₇₎ = −0.261, p = 0.802; licking: t₍₇₎ = −0.475, p = 0.649].

Figure 4

ChR2 stimulation of glutamatergic afferent fibers from the STN in the GP produced similar changes in c-Fos expression in the GP, M1, STN, EPN, and CPu as ChR2 stimulation of GABAergic neurons in the GP. (A) c-Fos expression in the light-exposed (right) and contralateral (left) hemisphere of the mouse after ChR2 stimulation of glutamatergic afferent fibers from the STN in the GP. c-Fos expression was detected in the GP, M1, STN, EPN, and CPu. (B) Quantitative analysis of c-Fos-positive cell counts in the GP, M1, STN, EPN, and CPu are presented in a bar graph. Asterisks indicate significant differences between the ipsilateral hemisphere and contralateral hemisphere to optical stimulation [***p < 0.001; n = 8, using a paired, two-tailed t-test; GP: t₍₇₎ = 11.143, p < 0.001; M1: t₍₇₎ = 14.697, p < 0.001; CPu: t₍₇₎ = 13.353, p < 0.001; EPN: t₍₇₎ = −16.253, p < 0.001; STN: t₍₇₎ = 0.607, p = 0.563].