Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Journal name:
Nature
Volume:
466,
Pages:
622–626
Date published:
DOI:
doi:10.1038/nature09159
Received
Accepted
Published online

Neural circuits of the basal ganglia are critical for motor planning and action selection1, 2, 3. Two parallel basal ganglia pathways have been described4, and have been proposed to exert opposing influences on motor function5, 6, 7. According to this classical model, activation of the ‘direct’ pathway facilitates movement and activation of the ‘indirect’ pathway inhibits movement. However, more recent anatomical and functional evidence has called into question the validity of this hypothesis8, 9, 10. Because this model has never been empirically tested, the specific function of these circuits in behaving animals remains unknown. Here we report direct activation of basal ganglia circuitry in vivo, using optogenetic control11, 12, 13, 14 of direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of channelrhodopsin-2 in the striatum of bacterial artificial chromosome transgenic mice expressing Cre recombinase under control of regulatory elements for the dopamine D1 or D2 receptor. Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. In a mouse model of Parkinson’s disease, direct-pathway activation completely rescued deficits in freezing, bradykinesia and locomotor initiation. Taken together, our findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behaviour and indicate that modulation of direct-pathway circuitry may represent an effective therapeutic strategy for ameliorating parkinsonian motor deficits.

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Figures

  1. Selective viral-mediated ChR2 expression in striatal direct- or indirect-pathway MSNs.
    Figure 1: Selective viral-mediated ChR2 expression in striatal direct- or indirect-pathway MSNs.

    a, Schematic of the double-floxed Cre-dependent AAV vector expressing ChR2–YFP under control of the EF-1α promoter. DIO, double-floxed inverted open reading frame; eYFP, enhanced YFP; ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. b, Sagittal mouse brain schematic. Ctx, cortex; Str, striatum; GP, globus pallidus; SNr, substantia nigra pars reticulata; Th, thalamus; Hipp, hippocampus. The box indicates the region shown in panels c and d. c, Sagittal section showing striatal direct-pathway MSNs expressing ChR2–YFP following injection of Cre-dependent AAV1 into D1-Cre BAC transgenic mice. Direct-pathway MSN axons target the SNr. d, Expression of ChR2–YFP in striatal indirect-pathway MSNs of D2-Cre BAC transgenic mice. Indirect-pathway MSN axons target the GP. Scale bars in c and d, 1mm. e, f, Examples of ChR2–YFP-expressing neurons that do not co-express the interneuronal markers parvalbumin (PV) and choline acetyltransferase (ChAT). Scale bars in e and f, 15μm. g, Percent of ChAT, PV or neuropeptide Y neurons that co-express ChR2–YFP. Error bars, s.e.m.

  2. ChR2-mediated excitation of direct- and indirect-pathway MSNs in vivo drives activity in basal ganglia circuitry.
    Figure 2: ChR2-mediated excitation of direct- and indirect-pathway MSNs in vivo drives activity in basal ganglia circuitry.

    a, Whole-cell current-clamp recordings from ChR2–YFP-positive neurons in vitro demonstrate normal current–firing relationships consistent with direct-pathway (red traces) and indirect-pathway (green traces) MSNs (D1-control, n = 10; D1-ChR2, n = 3; D2-control, n = 7; D2-ChR2, n = 3). b, Firing rate plotted as a function of injected current in direct- and indirect-pathway MSNs expressing either green fluorescent protein or ChR2–YFP. c, ChR2-mediated photocurrents (top) and spiking (bottom) in direct-pathway (left) and indirect-pathway (right) MSNs. In this and subsequent panels, blue bars indicate illumination time. d, Summary of ChR2-mediated photocurrents (left) and spiking (right) for D1-ChR2 (n = 5) and D2-ChR2 (n = 4) cells. e, Schematic of in vivo optical stimulation and recording in the striatum. f, Example MSN recorded from the striatum of an anaesthetized D1-ChR2 mouse that showed increased firing in response to illumination. Insets in f, g, j and k show spike waveforms with (blue) or without (grey) illumination. Scale bars apply to insets in f, g, j and k. g, Example of a light-sensitive MSN from a D2-ChR2 mouse. h, Normalized change in MSN firing rates in response to striatal illumination in D1-ChR2 (n = 16) and D2-ChR2 (n = 10) mice. Pre, before illumination; laser, during illumination; post, after illumination. i, Schematic of in vivo optical stimulation in striatum and recording in SNr. j, Example of a SNr neuron recorded from a D1-ChR2 mouse that was inhibited by direct-pathway activation. k, Example of a SNr neuron recorded from a D2-ChR2 mouse that was excited by indirect-pathway activation. l, Normalized change in SNr firing rate in response to activation of the direct (D1, n = 8) or indirect (D2, n = 4) pathways. *P<0.05; error bars, s.e.m.

  3. In vivo activation of direct or indirect pathways reveals pathway-specific regulation of motor function.
    Figure 3: In vivo activation of direct or indirect pathways reveals pathway-specific regulation of motor function.

    a, Coronal schematic of cannula placement and bilateral fibre-optic stimulation. b, Example of altered motor activity during bilateral striatal illumination in D1-ChR2 (left) and D2-ChR2 (right) mice. Lines represent the mouse’s path; dots represent the mouse’s location every 300ms. Grey paths represent 20s of activity before illumination; coloured paths represent 20s of activity during subsequent illumination. c, Motor activity before, during and after bilateral striatal illumination in D1-ChR2 (left, red) and D2-ChR2 (right, green) mice. di, Effect of illumination on the velocity of fine movements (d), initiation of ambulatory bouts (e), ambulation bout duration (f), ambulation velocity (g), frequency of freezing (h) and duration of freezing bouts (i) in D1-ChR2 (red bars, n = 9) and D2-ChR2 (green bars, n = 8) mice. j, No change in gait in response to illumination in D1-ChR2 (red bars, n = 4) or D2-ChR2 (green bars, n = 5) mice. *P<0.05; error bars, s.e.m.

  4. Direct-pathway activation rescues motor deficits in the 6-OHDA model of Parkinson/'s disease.
    Figure 4: Direct-pathway activation rescues motor deficits in the 6-OHDA model of Parkinson’s disease.

    a, Visualization of striatal dopaminergic afferents by tyrosine hydroxylase (TH) staining in coronal slices. Scale bar, 1mm. b, Loss of dopaminergic innervation in dorsomedial striatum one week after 6-OHDA injection. The arrow marks the injection site. c, ChR2–YFP expression in dorsomedial striatum of 6-OHDA-lesioned mice. d, Merged image shows overlap of ChR2 expression with the 6-OHDA lesion. e, Motor behaviour before (left, black bars) and after (right, red bars) 6-OHDA lesion in D1-ChR2 mice (n = 10). In 6-OHDA-lesioned mice, behaviour is shown before, during and after activation of the direct pathway. fk, Effect of 6-OHDA lesion and direct pathway rescue on fine-movement velocity (f), initiation of ambulatory bouts (g), ambulation bout duration (h), ambulation velocity (i), frequency of freezing (j) and duration of freezing bouts (k). l, No change in gait was observed after 6-OHDA lesioning or direct-pathway activation. *P<0.05; error bars, s.e.m.

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Author information

Affiliations

  1. Gladstone Institute of Neurological Disease, 1650 Owens St, San Francisco, California 94158, USA

    • Alexxai V. Kravitz,
    • Benjamin S. Freeze,
    • Philip R. L. Parker,
    • Kenneth Kay,
    • Myo T. Thwin &
    • Anatol C. Kreitzer
  2. Departments of Physiology and Neurology, University of California, San Francisco, California 94143, USA

    • Anatol C. Kreitzer
  3. Neuroscience Graduate Program, University of California, San Francisco, California 94158, USA

    • Philip R. L. Parker &
    • Anatol C. Kreitzer
  4. Biomedical Sciences Program, University of California, San Francisco, California 94143, USA

    • Benjamin S. Freeze &
    • Anatol C. Kreitzer
  5. Medical Scientist Training Program, University of California, San Francisco, California 94143, USA

    • Benjamin S. Freeze,
    • Kenneth Kay &
    • Anatol C. Kreitzer
  6. Departments of Bioengineering and Psychiatry and Behavioral Sciences, Stanford University, California 94305, USA

    • Karl Deisseroth

Contributions

A.V.K., P.R.L.P. and M.T.T. collected and processed tissue, and analysed immunohistochemical experiments. B.S.F. performed and analysed in vitro electrophysiology experiments. A.V.K., B.S.F. and P.R.L.P. performed and analysed in vivo electrophysiology experiments. A.V.K., P.R.L.P. and K.K. performed and analysed optogenetic/behavioural experiments. K.D. provided Cre-dependent ChR2–YFP and YFP-control constructs. A.C.K. designed and coordinated the study, and A.C.K. and A.V.K wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Information (1.1M)

    This file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1-2.

Movies

  1. Supplementary Movie 1 (22.5M)

    This movie shows the behavioral effect of unilateral stimulation of striatal indirect pathway MSNs on the left side. The text "LASER" appears in the upper left corner when the fiber is illuminated. Note the ipsiversive turning and immobility during laser illumination.

  2. Supplementary Movie 2 (19M)

    This movie shows the behavioral effect of bilateral stimulation of striatal indirect pathway MSNs. The text "LASER" appears in the upper left corner during laser illumination. Note the profound freezing during multiple trials of laser illumination.

  3. Supplementary Movie 3 (21.3M)

    This movie shows the behavioral effect of bilateral stimulation of the direct pathway in a 6-OHDA treated mouse. The text "LASER" appears in the upper left corner during laser illumination. Note the minimal movement and lack of rearing under basal (parkinsonian) conditions. The mouse has been bilaterally treated 5 days prior with 6-OHDA in the striatum to lesion dopamine projections. During direct pathway activation, the animal becomes more alert, moves more rapidly, and rears more often.

Additional data