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, 324 (5925), 354-9

Optical Deconstruction of Parkinsonian Neural Circuitry

Affiliations

Optical Deconstruction of Parkinsonian Neural Circuitry

Viviana Gradinaru et al. Science.

Abstract

Deep brain stimulation (DBS) is a therapeutic option for intractable neurological and psychiatric disorders, including Parkinson's disease and major depression. Because of the heterogeneity of brain tissues where electrodes are placed, it has been challenging to elucidate the relevant target cell types or underlying mechanisms of DBS. We used optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving parkinsonian rodents and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region. In addition to providing insight into DBS mechanisms, these results demonstrate an optical approach for dissection of disease circuitry and define the technological toolbox needed for systematic deconstruction of disease circuits by selectively controlling individual components.

Figures

Fig. 1.
Fig. 1.
Direct optical inhibition of local STN neurons. (A) Cannula placement, virus injection, and fiber depth were guided by recordings of the STN, which is surrounded by the silent zona incerta (ZI) and internal capsule (IC). (B) Confocal images of STN neurons expressing CaMKIIα::eNpHR-EYFP and labeled for excitatory neuron-specific CaMKIIα (right). (C) Continuous 561-nm illumination of the STN expressing CaMKIIα::eNpHR-EYFP in anesthetized 6-OHDA rats reduced STN activity; representative optrode trace and amplitude spectrum are shown. Mean spiking frequency was reduced from 29 ± 3 to 5 ± 1 Hz (means ± SEM, P < 0.001, Student’s t test, n = eight traces from different STN coordinates in two animals). (D) Amphetamine-induced rotations were not affected by stimulation of the STN in these animals (P > 0.05, n = 4 rats, t test with μ = 0). The red arrow indicates direction of pathologic effects; the green arrow indicates direction of therapeutic effects. The electrical control implanted with a stimulation electrode showed therapeutic effects with HFS (120 to 130 Hz, 60-μs pulse width, 130 to 200 μA, P < 0.05, t test with μ = 0). Percentage change of −100% indicates that the rodent is fully corrected. Data in all figures are means ± SEM ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.
Fig. 1.
Fig. 1.
Direct optical inhibition of local STN neurons. (A) Cannula placement, virus injection, and fiber depth were guided by recordings of the STN, which is surrounded by the silent zona incerta (ZI) and internal capsule (IC). (B) Confocal images of STN neurons expressing CaMKIIα::eNpHR-EYFP and labeled for excitatory neuron-specific CaMKIIα (right). (C) Continuous 561-nm illumination of the STN expressing CaMKIIα::eNpHR-EYFP in anesthetized 6-OHDA rats reduced STN activity; representative optrode trace and amplitude spectrum are shown. Mean spiking frequency was reduced from 29 ± 3 to 5 ± 1 Hz (means ± SEM, P < 0.001, Student’s t test, n = eight traces from different STN coordinates in two animals). (D) Amphetamine-induced rotations were not affected by stimulation of the STN in these animals (P > 0.05, n = 4 rats, t test with μ = 0). The red arrow indicates direction of pathologic effects; the green arrow indicates direction of therapeutic effects. The electrical control implanted with a stimulation electrode showed therapeutic effects with HFS (120 to 130 Hz, 60-μs pulse width, 130 to 200 μA, P < 0.05, t test with μ = 0). Percentage change of −100% indicates that the rodent is fully corrected. Data in all figures are means ± SEM ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.
Fig. 2.
Fig. 2.
Targeting astroglia within the STN. (A) Confocal images show STN astrocytes expressing GFAP::ChR2-mCherry,costainedwithGFAP (right). (B) A 473-nm illumination of the STN expressing GFAP::ChR2-mCherry inanesthetized6-OHDA rats. Optrode recording revealed that continuous illumination inhibited STN activity with delay to onset of 404 ± 39 ms and delay to offset of 770 ± 82 ms (n = five traces from different STN coordinates in two animals), nevertheless, the 50% duty cycle also inhibited spiking, with delay to onset of 520 ± 40 ms and delay to offset of 880 ± 29 ms (n = three traces from different STN coordinates in two animals) with P <0.001. (C) Amphetamine-induced rotations were not affected by 50% duty cycle illumination in these animals (right, P > 0.05, n = seven rats, t test with μ = 0).
Fig. 2.
Fig. 2.
Targeting astroglia within the STN. (A) Confocal images show STN astrocytes expressing GFAP::ChR2-mCherry,costainedwithGFAP (right). (B) A 473-nm illumination of the STN expressing GFAP::ChR2-mCherry inanesthetized6-OHDA rats. Optrode recording revealed that continuous illumination inhibited STN activity with delay to onset of 404 ± 39 ms and delay to offset of 770 ± 82 ms (n = five traces from different STN coordinates in two animals), nevertheless, the 50% duty cycle also inhibited spiking, with delay to onset of 520 ± 40 ms and delay to offset of 880 ± 29 ms (n = three traces from different STN coordinates in two animals) with P <0.001. (C) Amphetamine-induced rotations were not affected by 50% duty cycle illumination in these animals (right, P > 0.05, n = seven rats, t test with μ = 0).
Fig. 3.
Fig. 3.
Optical depolarization of STN neurons at different frequencies. (A) Confocal images of STN neurons expressing CaMKIIα::ChR2-mCherry and labeled for the excitatory neuron–specific CaMKIIα marker. (B) Optical HFS (120 Hz, 5-ms pulse width) of the STN expressing CaMKIIα::ChR2-mCherry in 6-OHDA rats recorded with the optrode connected to a 473-nm laser diode (representative trace and amplitude spectrum shown). Frequency of spiking increased from 41 ± 2 Hz to 85 ± 2 Hz (HFS versus pre, n = five traces: P <0.001, t test, post, n = three traces; traces were sampled from different STN coordinates in one animal). (C) Amphetamine-induced rotations were not affected by high (left, 130 Hz, 5-ms pulse width, n = five rats) or low (middle, 20 Hz, 5-ms pulse width, n = two rats) frequency optical stimulation.
Fig. 3.
Fig. 3.
Optical depolarization of STN neurons at different frequencies. (A) Confocal images of STN neurons expressing CaMKIIα::ChR2-mCherry and labeled for the excitatory neuron–specific CaMKIIα marker. (B) Optical HFS (120 Hz, 5-ms pulse width) of the STN expressing CaMKIIα::ChR2-mCherry in 6-OHDA rats recorded with the optrode connected to a 473-nm laser diode (representative trace and amplitude spectrum shown). Frequency of spiking increased from 41 ± 2 Hz to 85 ± 2 Hz (HFS versus pre, n = five traces: P <0.001, t test, post, n = three traces; traces were sampled from different STN coordinates in one animal). (C) Amphetamine-induced rotations were not affected by high (left, 130 Hz, 5-ms pulse width, n = five rats) or low (middle, 20 Hz, 5-ms pulse width, n = two rats) frequency optical stimulation.
Fig. 4.
Fig. 4.
Quantification of the tissue volume recruited by optical intervention. (A) Intensity values for 473-nm (blue) and 561-nm (yellow) light are shown for a 400-μm fiber as a function of depth in brain tissue. The dashed line at 1 mW/mm2 (30 mW light source) indicates the minimum intensity required to activate channelrhodopsins and halorhodopsins (16, 20). (B) Confocal images of STN neurons expressing CaMKIIα::ChR2-mCherry and labeled for the immediate early gene product c-fos show robust neuronal activation produced by light stimulation in vivo. Arrowheads indicate c-fos–positive cells. Freely moving rats expressing ChR2 in STN (same animals as in Fig. 3), were stimulated with 473-nm light (20 Hz, 5-ms pulse width). (C) The STN volume that showed strong c-fos activation was estimated to be at least 0.7 mm3 (dashed lines indicate STN boundaries); robust c-fos activation was observed medial-lateral (1.155 mm), anterior-posterior (0.800 mm), and dorsal-ventral (0.770 mm) on subthalamic slices imaged by confocal microscopy with 4′,6′-diamidino-2-phenylindole (DAPI) counterstain.
Fig. 4.
Fig. 4.
Quantification of the tissue volume recruited by optical intervention. (A) Intensity values for 473-nm (blue) and 561-nm (yellow) light are shown for a 400-μm fiber as a function of depth in brain tissue. The dashed line at 1 mW/mm2 (30 mW light source) indicates the minimum intensity required to activate channelrhodopsins and halorhodopsins (16, 20). (B) Confocal images of STN neurons expressing CaMKIIα::ChR2-mCherry and labeled for the immediate early gene product c-fos show robust neuronal activation produced by light stimulation in vivo. Arrowheads indicate c-fos–positive cells. Freely moving rats expressing ChR2 in STN (same animals as in Fig. 3), were stimulated with 473-nm light (20 Hz, 5-ms pulse width). (C) The STN volume that showed strong c-fos activation was estimated to be at least 0.7 mm3 (dashed lines indicate STN boundaries); robust c-fos activation was observed medial-lateral (1.155 mm), anterior-posterior (0.800 mm), and dorsal-ventral (0.770 mm) on subthalamic slices imaged by confocal microscopy with 4′,6′-diamidino-2-phenylindole (DAPI) counterstain.
Fig. 5.
Fig. 5.
Selective optical control of afferent fibers in the STN. (A) Confocal images of Thy1::ChR2-EYFP expression in the STN and DAPI staining for nuclei shows selective expression in fibers and not cell bodies (right). (B) Optical HFS (130 Hz, 5-ms pulse width) of the STN region in an anesthetized Thy1::ChR2-EYFP 6-OHDA mouse with 473-nm light inhibited STN large-amplitude spikes (sample trace, top left), while inducing smaller-amplitude high-frequency oscillations (figs. S4, C and D, and S5C). Optical LFS (20 Hz, 5-ms pulse width) produced reliable spiking at 20 Hz (bottom left). Whereas HFS prevented bursting (top right, P <0.001, n = 3), LFS had no significant effect on burst frequency by two-sample t test (P > 0.05, n = three traces) nor on spikes per burst (bottom right, P > 0.05, n = three traces). (C) Optical HFS to STN in these animals (left, 100 to 130 Hz, 5-ms pulse width, n = five mice) produced robust therapeutic effects, reducing ipsilateral rotations and allowing animals to freely switch directions. In contrast, optical LFS (second left, 20 Hz, 5-ms pulse width, n = five mice) exacerbated pathologic effects, causing increased ipsilateral rotations. Both effects were reversible (post). Changes were significant by t test with μ = 0 for both HFS (P < 0.001, n = five mice) and LFS (P < 0.05, n = five mice) compared with baseline (light off). (Right) Contralateral head position bias also showed robust correction with HFS by two-sample t test (HFS versus light off: P < 0.05; n = two mice), but not with LFS (LFS versus light off: P > 0.05, n = two mice).
Fig. 6.
Fig. 6.
Selective optical stimulation of layer V neurons in anterior primary motor cortex. (A) GAD67 and GABA staining showed no colocalization with Thy1::ChR2-EYFP in STN (left). Apical dendrites of layer V neurons can be seen rising to the pial surface (22, 23) (right). (B) Schematic for optical stimulation of M1 with simultaneous recording in STN of Thy1::ChR2 mice. (C) Optical stimulation (473 nm) of M1 and simultaneous recording in STN of anesthetized Thy1::ChR2 mice. Optical HFS (130 Hz, 5-ms pulse width) of M1 modulated activity in both M1 and STN. Optical LFS (20 Hz, 5-ms pulse width) of M1 produced 20-Hz tonic firing in both M1 and STN. (D) Optical HFS (130 Hz, 5-ms pulse width) reduced amphetamine-induced ipsilateral rotations in 6-OHDA Thy1::ChR2 mice (P < 0.01, n = five mice) in contrast to optical LFS (20 Hz, 5-ms pulse width, P > 0.05, n = four mice); t test with μ = 0. (E) Contralateral head position bias was corrected in HFS (HFS versus light off: P < 0.001, n = four mice), whereas LFS had little effect (LFS versus light off: P > 0.05, n = three mice); two-sample t test. (F) HFS but not LFS to M1 significantly increased path length (HFS, P < 0.01, n = two mice) and climbing (P < 0.05, n = three mice); two-sample t test. Sample paths before, during, and after HFS are shown (100 s each, path lengths noted in cm).

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