Asymmetric cortical projections to striatal direct and indirect pathways distinctly control actions
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, United States
- New Cornerstone Science Laboratory, Center for Motor Control and Disease, Key Laboratory of Brain Functional Genomics, East China Normal University, China
- Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, United States
- NYU–ECNU Institute of Brain and Cognitive Science, New York University Shanghai, China
Figures
Figure 1 with 1 supplement
Selective labeling and functional expression of ChR2 in specific cortical neurons projecting to striatal D1- vs. D2-SPNs.
(A) Schematic of SAD-ΔG-Rabies-ChR2-mCherry construct with the glycoprotein deleted and replaced with ChR2-mCherry. (B) Timeline of viral injections of Cre-dependent helper viruses and the modified rabies virus for slice and behavioral experiments. (C) Example of coronal brain section with rabies-eGFP injection in the dorsal medial striatum of D1-Cre (top) or A2a-Cre (bottom) mouse shows enriched eGFP expression in the MCC. Scale bar, 1 mm. Inset (right): Higher magnification of retrogradely labeled striatal D1- or D2-SPN projecting neurons in the MCC expressing eGFP. Dotted lines demarcate cortical lamina. Scale bar, 200 µm. (D) Similar experiments of labeling striatal D1- vs. D2-SPN projecting neurons in M1 with rabies-eGFP. (E) Cartoon brain schematic of ChR2-mCherry expressing M1 neurons projecting to SPNs (red) during whole-cell patch clamp recordings. (F) Example of coronal brain section with rabies-ChR2-mCherry injection in the dorsal lateral striatum of A2a-Cre mouse. Scale bar, 1 mm. Inset (right): Higher magnification of retrogradely labeled striatal D2-SPN projecting neurons in the M1 showed clear membrane expression of ChR2-mCherry. Scale bar, 200 µm. (G) (left) 10× epifluorescent (red channel) of ChR2-mCherry-positive neurons in M1. Scale bar, 250 µm. (middle) 40× image of a patched layer 5 pyramidal neuron under DIC optics. Scale bar, 50 µm. (right) Epifluorescent image (red channel) showing patched layer 5 pyramidal neuron somas expressing ChR2-mCherry signal. Red dotted line denotes patched neuron. Scale bar, 50 µm. (H) Current-voltage traces of a ChR2-mCherry positive layer 5 M1 neuron under current clamp responding to hyperpolarizing and depolarizing current injection steps. Scale bars, 200 ms, 25 mV. (I) Optogenetic stimulation (20 Hz) elicits robust action potentials with high fidelity in a ChR2-mCherry positive D1-SPN projecting M1 neuron in layer 5. Scale bars, 100 ms, 20 mV.
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Figure 1—source data 1
Current-clamp recording of a rabies-labeled cortical neuron expressing ChR2-mCherry.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig1-data1-v1.pptx
Figure 1—figure supplement 1
Optogenetic stimulation at 5 Hz reliably evoked action potential firing in a ChR2-expressing M1 neuron labeled by rabies infection.
5 Hz optogenetic stimulation elicits action potentials with high fidelity in a ChR2-mCherry positive M1 pyramidal neuron in layer 5 projecting to striatal D1-SPNs. Scale bars, 200 ms, 25 mV.
Figure 2 with 1 supplement
The excitatory inputs to striatal D1- vs. D2-SPNs are partially segregated with asymmetrical overlapping.
(A) Schematic for the possible organization of the excitatory inputs to striatal D1- vs. D2-SPNs from completely segregated (left), totally overlapping (middle), to partially mixed (right). The red and black filled circles indicate the individual neurons projecting to D1- vs. D2-SPNs, respectively. The half red and half black circles imply the neurons projecting to both. (B) (left) Schematic of rabies-ChR2 labeling of the inputs to D1-SPNs and whole-cell recordings of rabies-negative striatal D1-SPNs with local optogenetic stimulation. (right) Example of the average EPSC trace showing short latency response to paired pulses (50 ms ISI) stimulation (black), that is blocked by AMPAR and NMDAR antagonists (gray). All recordings were conducted in the presence of picrotoxin (PTX) to isolate excitatory transmission. Scale bar, 25 ms, 100 pA. Same conditions applied to all following recordings. (C) Whole-cell recording of rabies-negative striatal D2-SPNs with local optogenetic stimulation with rabies-ChR2 labeling of the inputs to D2-SPNs. (D) The likelihood of the inputs to D1-SPNs forming a functional connection with nearby non-starter D1-SPNs, and the likelihood of the D2-SPN situation. Numbers above the bars denote the number of cells that show functional connectivity within total recorded. Fisher’s exact test, p=0.3137. Number of animals in each group: D1-projecting to D1 EGFP(+), N=7; D1-projecting to D2 EGFP(−), N=8; D2-projecting to D2 EGFP(+), N=10; D2-projecting to D1 EGFP(−), N=8. (E–F) Whole-cell recording of rabies-negative striatal D2-SPNs with local optogenetic stimulation with rabies-ChR2 labeling of the inputs to D1-SPNs (E), and recording of rabies-negative D1-SPNs with stimulation of inputs to D2-SPNs (F). (G) The likelihood of the inputs to D1-SPNs forming a functional connection with nearby non-starter D2-SPNs, and the likelihood of the inputs to D2-SPNs forming a functional connection with nearby non-starter D1-SPNs. Fisher’s exact test, p=0.0079. **, p<0.01. Number of animals in each group: D1-projecting to D2 EGFP(+), N=8; D1-projecting to D1 EGFP(−), N=7; D2-projecting to D1 EGFP(+), N=8; D2-projecting to D2 EGFP(−), N=10.
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Figure 2—source data 1
Number of connections from D1- or D2-projecting cortical neurons onto non-starter D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The synaptic properties of projections from D1- or D2-SPN retrogradely labeled cortical inputs to striatal D1- or D2-SPNs.
(A–D) The EPSC latency (A), amplitudes (B), paired pulse ratio (C), and variation (D) of whole-cell recordings of rabies-negative striatal D1- or D2-SPNs, with optogenetic stimulation of the terminals of D1- or D2-SPN retrogradely labeled cortical neurons. n.s., p>0.05, not statistically significant. Data are expressed as mean ± SEM.
Figure 3 with 2 supplements
Different effects of optogenetic stimulation of D1- vs. D2-SPN projecting cortical neurons on locomotion and reinforcement learning.
(A) Schematic of largely segregated yet partially overlapping excitatory inputs to striatal D1- vs. D2-SPNs. (B) Schematic of dorsal medial striatum (DMS) injection of Cre-dependent AAV-ChR2 and optogenetic stimulation in D1- or A2a-Cre mice. (C) (top) Example of locomotion path under control (black) and following 20 Hz optogenetic stimulation (gray) of DMS D1-SPNs in open field. Scale bar, 5 cm, same for below. (bottom) Stimulation of D1-SPNs in DMS facilitates locomotion (n=5, unpaired two-tailed t-test, t=3.386, p=0.0046). **, p<0.01. Data are expressed as mean ± SEM. (D) 20 Hz stimulation of D2-SPNs in DMS suppresses locomotion (n=5, unpaired two-tailed t-test, t=2.559, p=0.0227). *, p<0.05. Data are expressed as mean ± SEM. (E) Schematic for dorsal lateral striatum (DLS) optogenetics. (F–G) 20 Hz stimulation of D1-SPNs in DLS facilitates locomotion (F, n=5, unpaired two-tailed t-test, t=4.736, p=0.0003), while stimulation of D2-SPNs in DLS does not significantly suppress locomotion in open field (G, n=5, unpaired two-tailed t-test, t=1.026, p=0.3224). ***, p<0.001. Data are expressed as mean ± SEM. (H) Schematic of rabies-ChR2 labeling of the inputs to D1 or D2-SPNs and optogenetic stimulation in MCC. (I–J) 20 Hz stimulation of MCC neurons projecting to D1-SPNs facilitates locomotion (I, n=9, unpaired two-tailed t-test, t=2.344, p=0.0344), while stimulation of MCC neurons projecting to D2-SPNs does not alter locomotion (J, n=10, unpaired two-tailed t-test, t=1.214, p=0.2447). *, p<0.05. Data are expressed as mean ± SEM. (K) Schematic of rabies-ChR2 labeling of the inputs to D1 or D2-SPNs and optogenetic stimulation in M1. (L–M) 20 Hz stimulation of the M1 neurons projecting to D1-SPNs facilitates locomotion (L, n=7, unpaired two-tailed t-test, t=3.276, p=0.0055), while stimulation of the M1 neurons projecting to D2-SPNs does not significantly alter locomotion (M, n=8, Unpaired two-tailed t-test, t=0.5796, p=0.5714). **, p<0.01. Data are expressed as mean ± SEM. (N) Schematic of a mouse performing intracranial self-stimulation (ICSS) behavior. (O–P) D1-SPN (red) but not D2-SPN stimulation (black) drives ICSS behavior in both the DMS (O: D1, n=6, permutation test, slope = 1.5060, p=0.0378; D2, n=5, permutation test, slope = –0.2214, p=0.1021; one-tailed Mann Whitney test, Day 7 D1 vs. D2, p=0.0130) and the DLS (P: D1, n=6, permutation test, slope = 28.1429, p=0.0082; D2, n=5, permutation test, slope = –0.3429, p=0.0463; one-tailed Mann Whitney test, Day 7 D1 vs. D2, p=0.0390). *, p<0.05. Data are expressed as mean ± SEM. (Q) Timeline of helper virus injections, rabies-ChR2 injections and optogenetic stimulation for ICSS behavior. (R–S) Optogenetic stimulation of the cortical neurons projecting to either D1- or D2-SPNs induces ICSS behavior in both the MCC (R: MCC-D1, n=5, permutation test, Day1-Day7, slope = 2.5857, p=0.0034; MCC-D2, n=5, Day2-Day7, permutation test, slope = 1.4229, p=0.0344; no significant effect on Day7, MCC-D1 vs. MCC-D2, two-tailed Mann Whitney test, p=0.9999) and the M1 (S: M1-D1, n=5, permutation test, Day1-Day7, slope = 1.8214, p=0.0259; M1-D2, n=5, Day1-Day7, permutation test, slope = 1.8214, p=0.0025; no significant effect on Day7, M1-D1 vs. M1-D2, two-tailed Mann-Whitney test, p=0.3810). n.s., not statistically significant. Data are expressed as mean ± SEM.
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Figure 3—source data 1
Effect of 20 Hz optogenetic stimulation on normalized distance traveled for D1- or D2-SPNs in DMS and DLS.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-data1-v1.xlsx
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Figure 3—source data 2
Normalized distance traveled during 20 Hz optogenetic stimulation of MCC neurons projecting to D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-data2-v1.xlsx
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Figure 3—source data 3
Normalized distance traveled during 20 Hz optogenetic stimulation of M1 neurons projecting to D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-data3-v1.xlsx
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Figure 3—source data 4
ICSS behavior induced by 20 Hz optogenetic stimulation of D1- or D2-SPNs in the DMS and DLS.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-data4-v1.xlsx
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Figure 3—source data 5
ICSS behavior induced by 20 Hz optogenetic stimulation of D1- or D2-projecting neurons in the MCC and M1.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-data5-v1.xlsx
Figure 3—figure supplement 1
Low-frequency (5 Hz) optogenetic stimulation of cortical neurons projecting to striatal D1- or D2-SPNs has little effect on locomotion activity.
(A–B) 5 Hz optogenetic stimulation on MCC neurons projecting to either D1- or D2-SPNs didn’t change the locomotion activity. MCC – D1, n=9, unpaired two-tailed t-test, t=0.1906, p=0.8516. MCC – D2, n=10, unpaired two-tailed t-test, t=1.015, p=0.3275. Data are expressed as mean ± SEM. (C) 5 Hz optogenetic stimulation of M1 neurons projecting to D1-SPNs didn’t change the locomotion activity. n=7, unpaired two-tailed t-test, t=0.276, p=0.7866. Data are expressed as mean ± SEM. (D) 5 Hz optogenetic stimulation of M1 neurons projecting to D2-SPNs slightly increased locomotion activity. n=8, unpaired two-tailed t-test, t=2.48, p=0.0265. *, p<0.05. Data are expressed as mean ± SEM.
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Figure 3—figure supplement 1—source data 1
Normalized distance traveled during 5 Hz optogenetic stimulation of MCC neurons projecting to D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
Normalized distance traveled during 5 Hz optogenetic stimulation of M1 neurons projecting to D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-figsupp1-data2-v1.xlsx
Figure 3—figure supplement 2
No effects of optogenetic stimulation of M1 on locomotion in mice with ChR2 expression in either D1- or D2-SPNs of DMS.
(A) Schematic of dorsal medial striatum (DMS) injection of Cre-dependent AAV-ChR2 in D1- and A2a-Cre mice with optogenetic stimulation in M1. (B) 20 Hz optogenetic stimulation of M1 in mice expressing ChR2 in striatal D1-SPNs didn’t change the locomotion activity. n=5, unpaired two-tailed t-test, t=0.1016, p=0.9194. Data are expressed as mean ± SEM. (C) 20 Hz optogenetic stimulation of M1 in mice expressing ChR2 in striatal D2-SPNs didn’t alter the locomotion activity. n=5, unpaired two-tailed t-test, t=1.155, p=0.2525. Data are expressed as mean ± SEM.
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Figure 3—figure supplement 2—source data 1
Normalized distance traveled during 20 Hz optogenetic stimulation of M1 with ChR2 expressed exclusively in striatal D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig3-figsupp2-data1-v1.xlsx
Figure 4
Optogenetic stimulation of D1- vs. D2-SPN projecting cortical neurons differently modulates action sequence execution.
(A) Schematic of a mouse performing FR8 sequence. (B) Optogenetic stimulation (20 Hz) of the D1-SPN projecting MCC neurons during FR8 sequence. Example lever pressing (black bar) in control (top) vs. stimulation (middle) trials aligned to the first press, where the blue transparent rectangle corresponds to the window of optogenetic stimulation (20 Hz, 8 s). The black and blue lines in the PETH (bottom) indicate the lever press rate for control and stimulation conditions, respectively, same for below. (C) Optogenetic stimulation (20 Hz) of the D2-SPN projecting MCC neurons during FR8 sequence. (D) Average percent lever press rate change during optogenetic stimulation of D1- vs. D2-SPN projecting MCC neurons compared to control (MCC – D1, n=8; MCC – D2, n=7; Unpaired two-tailed t-test, t=2.774, p=0.0097). **, p<0.01. Effects compared to a theoretical percentage change of 0 of each individual manipulation (MCC-D1, n=8, one-sample two-tailed t-test, t=2.814, p=0.0131, 95% CI, 15.49–112.2; MCC-D2, n=7, one-sample two-tailed t-test, t=0.8481, p=0.4117, 95% CI, –21.78–9.502). Data are expressed as mean ± SEM. (E) Timeline of helper virus injections, rabies-ChR2 injections and optogenetic stimulation during action sequence performance. (F–G) Optogenetic stimulation (20 Hz) of the D1- (F) or D2-SPN (G) projecting M1 neurons during FR8 sequence. (H) Average percent lever press rate change during optogenetic stimulation of D1- vs. D2-SPN projecting M1 neurons compared to control (M1 – D1, n=6; M1 – D2, n=7; Unpaired two-tailed t-test, t=0.7651, p=0.4511). Effects compared to a theoretical percentage change of 0 of each individual manipulation (M1-D1, n=6, one-sample two-tailed Wilcoxon signed-rank test, p=0.0046, 97.75% CI, –0.7866–151.0; M1-D2, n=7, one-sample two-tailed Wilcoxon signed-rank test, p=0.0479, 96.48% CI, 2.350–62.86). Data are expressed as mean ± SEM.
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Figure 4—source data 1
Change in sequence pressing during 20 Hz optogenetic stimulation of MCC or M1 neurons projecting to striatal D1- or D2-SPNs.
- https://cdn.elifesciences.org/articles/92992/elife-92992-fig4-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Drd1-Cre | The Jackson Laboratory | stock # 030329; RRID:IMSR_JAX:030329 | maintained on a C57BL/6 J background |
| Strain, strain background (Mus musculus) | Adora2a-Cre | The Jackson Laboratory | stock # 036158; RRID:MMRRC_036158-UCD | maintained on a C57BL/6 J background |
| Strain, strain background (Mus musculus) | C57BL/6 J | The Jackson Laboratory | stock # 000664; RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus) | D1-eGFP | MMRRC | MMRRC_000297-MU; RRID:MMRRC_000297-MU | GENSAT: X60 |
| Strain, strain background (Mus musculus) | D2-eGFP | MMRRC | MMRRC_00230-UNC; RRID:MMRRC_000230-UNC | GENSAT: S118 |
| Strain, strain background (Adeno-associated virus) | AAV5/EF1α-Flex-TVA-mCherry | UNC Viral Vector Core | RRID:SCR-002448 | 3–4.3×1012 particles/mL |
| Strain, strain background (Adeno-associated virus) | AAV8/CA-Flex-RG | UNC Viral Vector Core | RRID:SCR-002448 | 1.2–4.3×1012 particles/mL |
| Strain, strain background (Pseudotyped rabies virus) | (EnvA) SAD-∆G Rabies-eGFP | Salk Vector Core | RRID:SCR_014847 | |
| Strain, strain background (Pseudotyped rabies virus) | (EnvA) SAD-∆G Rabies-ChR2-mCherry | Salk Vector Core | RRID:SCR_014847 | |
| Strain, strain background (Adeno-associated virus) | AAV5-EF1α-DIO-ChR2(H134R)-mCherry | Salk Vector Core | RRID:SCR_014847 | |
| Chemical compound, drug | NBQX disodium salt hydrate | MilliporeSigma | Cat.#. N183 | 10 µM (final) |
| Chemical compound, drug | DL-APV | MilliporeSigma | Cat.#. A5282 | 50 µM (final) |
| Chemical compound, drug | Picrotoxin | MilliporeSigma | Cat.#. P1675 | 50–100 µM (final) |
| Chemical compound, drug | QX-314 | MilliporeSigma | Cat.#. L5783 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | Fiji / ImageJ | NIH | RRID:SCR_002285 | |
| Software, algorithm | pClamp 9.2 | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | Illustrator | Adobe | RRID:SCR_010279 | |
| Software, algorithm | MED PC | MED Associates | RRID:SCR_012156 | |
| Other | Allen Reference Atlas | RRID:SCR_013286 |
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Asymmetric cortical projections to striatal direct and indirect pathways distinctly control actions
eLife 12:RP92992.
https://doi.org/10.7554/eLife.92992.4
