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Cell-specific pallidal intervention induces long-lasting motor recovery in dopamine-depleted mice

Abstract

The identification of distinct cell types in the basal ganglia has been critical to our understanding of basal ganglia function and the treatment of neurological disorders. The external globus pallidus (GPe) is a key contributor to motor suppressing pathways in the basal ganglia, yet its neuronal heterogeneity has remained an untapped resource for therapeutic interventions. Here we demonstrate that optogenetic interventions that dissociate the activity of two neuronal populations in the GPe, elevating the activity of parvalbumin (PV)-expressing GPe neurons over that of Lim homeobox 6 (Lhx6)-expressing GPe neurons, restores movement in dopamine-depleted mice and attenuates pathological activity of basal ganglia output neurons for hours beyond stimulation. These results establish the utility of cell-specific interventions in the GPe to target functionally distinct pathways, with the potential to induce long-lasting recovery of movement despite the continued absence of dopamine.

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Figure 1: Global GPe stimulation does not rescue movement in DD mice.
Figure 2: Selective stimulation of PV-GPe neurons rescues movement persistently in DD mice.
Figure 3: Local response during PV-ChR2 stimulation directly inhibits other high-firing GPe neurons.
Figure 4: Selective suppression of Lhx6-GPe neurons rescues movement persistently in DD mice.
Figure 5: The induction of persistent behavioral rescue is cell-type specific.
Figure 6: PV-ChR2 and Lhx6-Arch reverse pathological bursting activity persistently.

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Acknowledgements

The authors thank V. Corbit (University of Pittsburgh) and T. Whalen (Carnegie Mellon University) for Matlab analysis scripts and B. Rogowski (Carnegie Mellon University) for surgical support and behavioral video editing. We also thank N. Kessaris (University of College London) and H. Zeng (Allen Institute) for their gifts of the Lhx6-iCre and Pvalb-2A-Cre mice, respectively. This work was supported by NIH grants F31 NS090745-01 (K.M.), F31 NS093944-01 (A.W.) and R00 NS076524, NSF grant DMS 1516288, and grants from the Brain and Behavior Research Foundation (National Alliance for Research on Schizophrenia and Depression Young Investigator Grant), the Parkinson's Disease Foundation, and the NIH Intramural Research Program.

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Contributions

K.J.M., K.T.Z., and K.H.L. performed behavioral experiments, including the histological verification, and with A.H.G. analyzed the data. K.J.M. and A.M.W. were responsible for the collection and analysis of the in vivo experiments. All authors discussed results and interpretations. K.J.M. and A.H.G. designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Aryn H Gittis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Behavioral and pathophysiological symptoms of bilateral DD are apparent within 3–5 d post-depletion.

(a) Schematic of bilateral DD in the medial forebrain bundle (MFB). (b) Quantification of immobility and bradykinesia induced by unilateral (Uni, n = 4) and bilateral (Bi, n = 51) depletions, as compared to dopamine-intact controls (Naive, n = 4). (*p < 0.02, **p < 0.001, Mann Whitney U). (c) Rasters of single units in the GPe of naïve vs. bilateral DD mice. Scale bar, 500 ms. (d) Box plots showing decreases in firing rates (Naive: 44.3 ± 2.6 Hz, n = 73 across 4 animals, versus Acute: 24.6 ± 1.6 Hz,n = 62 across 3 animals, H(1) = 29.775, **p < 0.001, Kruskal-Wallis H test) and (e) increases in coefficients of variation of the interspike intervals (CVISI) (Naïve: 0.63 ± 0.03 versus Acute: 0.80 ± 0.03, H(1) = 22.615, *p < 0.001, Kruskal-Wallis H test) following bilateral DD. Error bars, sem.

Supplementary Figure 2 Histological verification of TH immunoreactivity, viral expression and fiber placements for behavioral optogenetics in global manipulations.

(a) Representative images of striatal TH immunoreactivity in dorsal striatum of healthy dopamine intact tissue compared to a fully depleted bilateral animal. Scale Bar, 200 μm (b) Epifluorescent images of viral expression and fiber identification (yellow arrow) within the GPe. Scale Bar, 500 μm (c) Superimposed traces of viral expression across animals within hSyn-ChR2 (d) Epifluorescent images of viral expression and fiber identification (yellow arrow) with the dorsal striatum. Scale Bar, 500 μm (e) Superimposed traces of viral expression across animals within D1-ChR2 condition. Ctx = Cortex, Str = Striatum, GPe = globus pallidus externa.

Supplementary Figure 3 Histological verification of viral expression and fiber placements for behavioral optogenetics in cell-type manipulations.

(a) Epifluorescent images of viral expression and fiber identification (yellow arrow) within the GPe. Scale Bar, 500 μm (b-f) Superimposed traces of viral expression across animals within PV-ChR2, Lhx6-Arch, CAG-Arch, Lhx6-ChR2 and PV-Arch conditions.

Supplementary Figure 4 PV-ChR2 stimulation induces transient effects in partially and unilaterally depleted mice.

(a) Percentage of time spent in the immobile state before, during and after PV-ChR2 stimulation in lipopolysaccharide (LPS) injected mice. (b) Schematic representation of striatal location for TH analysis with epifluorescent image of a partial depletion of tyrosine hydroxylase. Scale Bar, 200 μm. (c) Quantification of TH levels, normalized to dopamine intact littermate and subsequent categorization into partially and fully depleted animals.(d) Percentage of time spent in the immobile state before, during, and after PV-ChR2 stimulation in partially depleted mice. (e) Overlay of immobility immediately before (pre), during (stim), and after (post) each light pulse. (f) Percent time spent immobile for each animal (grey x = pre, black circle = post) and degree of rescue (red line) as a function of TH remaining. Note sharp cut-off for induction of behavioral rescue at ~20% dopamine remaining. (g) Percentage of time spent in the immobile state before, during, and after PV-ChR2 stimulation in unilaterally depleted mice. (h) Overlay of immobility immediately before (pre), during (stim), and after (post) each light pulse. (i) Percent time spent immobile over the course of the full experimental trial. Error bars, sem.

Supplementary Figure 5 Optical identification using ChR2 and Arch and their corresponding firing properties and waveforms.

(a) Representative responses over 20 trials from a ChR2+ (putative PV) and CHR2- (putative non-PV) neuron responding to 5 ms optical pulses. Yellow bar denotes first significant bin as compared to baseline (b) Firing rate and coefficient of variation of the interspike interval (CVISI) for ChR2+ and ChR2- neurons in dopamine depleted (FR: p = 0.128, CVISI: p = 0.005, Mann Whitney U) (c) Extracellular waveform analysis of the peak-valley ratio and amplitude of individual units identified as ChR2+ (red, closed circles) and ChR2- (black, open circles). Inset: Average waveforms of ChR2+ and ChR2- (Note: nearly complete overlap). Scale bar: 50 μV(vertical), 220 μsec (horizontal) (d) Firing rate and coefficient of variation of the interspike interval (CVISI) for ChR2+ and ChR2- neurons in dopamine intact animals (Naïve) (e) Representative responses over 20 trials from a Arch+ (putative Lhx6) and Arch- (putative non-Lhx6) neuron responding to 1 s optical pulses. (f) Firing rate and CVISI for Arch+ and Arch- neurons (FR: p = 0.990, CV: p = 0.454, Mann Whitney U). (g) Extracellular waveform analysis of the peak-valley ratio and amplitude of individual units identified as Arch+ (blue, closed squares) and Arch- (black, open squares). Inset: Average waveforms of Arch+ and Arch-. Scale bar: 50 μV(vertical), 220 μsec (horizontal).

Supplementary Figure 6 PV and Lhx6 overlap partially in the Lhx6-Cre transgenic mouse.

(a) Fluorescent images from the GPe showing overlap between Lhx6-iCre and PV. Left: Lhx6-EYFP neurons. Arrows denote position of PV+ neurons (Purple arrows: Lhx6/PV double labeled; Red arrows: PV-only neurons). Note weaker Lhx6-EYFP expression in Lhx6/PV neurons. Middle: PV+ neurons (Blue arrows: Lhx6-only neurons). Right: Overlay. (b) Box diagram summarizing the proportion of GPe neurons (n = 4194 total neurons across 5 animals) counted that expressed either PV, Lhx6, or both.

Supplementary Figure 7 SNr firing rate is unaltered after PV-ChR2 and Lhx6-Arch, but decreased after hSyn-ChR2 manipulation.

(a) Firing rate of single units collected before (pre) and after (post) stimulation in PV-ChR2 (nPre = 80 vs nPost = 58 units across 3 animals, p = 0.976, Mann Whitney U), hSYn-ChR2 (nPre = 55 vs nPost = 69 unit across 3 animals, p = 0.002, Mann Whitney U) and Lhx6-Arch (nPre = 30 vs nPost = 69 units across 3 animals, p = 0.142, Mann Whitney U).

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Mastro, K., Zitelli, K., Willard, A. et al. Cell-specific pallidal intervention induces long-lasting motor recovery in dopamine-depleted mice. Nat Neurosci 20, 815–823 (2017). https://doi.org/10.1038/nn.4559

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