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Locomotion activates PKA through dopamine and adenosine in striatal neurons

Abstract

The canonical model of striatal function predicts that animal locomotion is associated with the opposing regulation of protein kinase A (PKA) in direct and indirect pathway striatal spiny projection neurons (SPNs) by dopamine1,2,3,4,5,6,7. However, the precise dynamics of PKA in dorsolateral SPNs during locomotion remain to be determined. It is also unclear whether other neuromodulators are involved. Here we show that PKA activity in both types of SPNs is essential for normal locomotion. Using two-photon fluorescence lifetime imaging8,9,10 of a PKA sensor10 through gradient index lenses, we measured PKA activity within individual SPNs of the mouse dorsolateral striatum during locomotion. Consistent with the canonical view, dopamine activated PKA activity in direct pathway SPNs during locomotion through the dopamine D1 receptor. However, indirect pathway SPNs exhibited a greater increase in PKA activity, which was largely abolished through the blockade of adenosine A2A receptors. In agreement with these results, fibre photometry measurements of an adenosine sensor11 revealed an acute increase in extracellular adenosine during locomotion. Functionally, antagonism of dopamine or adenosine receptors resulted in distinct changes in SPN PKA activity, neuronal activity and locomotion. Together, our results suggest that acute adenosine accumulation interplays with dopamine release to orchestrate PKA activity in SPNs and proper striatal function during animal locomotion.

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Fig. 1: In vivo PKA activity imaging reveals cell-type-specific modulation of PKA by dopamine.
Fig. 2: Locomotion requires PKA activity and increases PKA activity in both dSPNs and iSPNs.
Fig. 3: A2A receptors mediate PKA activity increases in iSPNs.
Fig. 4: Locomotion results in acute adenosine accumulation.

Data availability

Original raw data will be provided upon request to include all supporting information. Source data are provided with this paper.

Code availability

The following custom codes have been made available at GitHub: SI_View (https://github.com/HZhongLab/SI_View; https://doi.org/10.5281/zenodo.6982316); FLIMview (https://github.com/HZhongLab/FLIMview; https://doi.org/10.5281/zenodo.6982385) and AnimalStateTracker (https://github.com/HZhongLab/animalStateTracker; https://doi.org/10.5281/zenodo.6984502).

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Acknowledgements

We thank J. Williams and all members of the Zhong and Mao laboratories for critical discussions throughout the project; R. Yasuda at Max Planck Florida for the FLIMimage software; S. Wang at Stanford University for the generation of the FLEX-tAKARα AAV; G. Mandel at Vollum for providing the rotarod and open-field setup; D.-T. Lin at the NIH for providing the Drd1a-Cre mouse line; Y. Li at Peking University for providing the GRABAdo viruses; J. Melander for early pilot experiments; T. L. Yiu for assistance in the behaviour hardware; Y. Chen for assistance in schematic illustrations; and J. Williams, M. Wolf and B. Jongbloets for critical comments on the manuscript. This work was supported by two BRAIN Initiative awards (NIH, USA; U01NS094247 to H.Z. and T.M., and R01NS104944 to H.Z. and T.M.), two R01 grants (R01NS081071 to T.M. and R01NS127013 to H.Z.) and an R21 grant (R21NS097856 to H.Z.), with the latter three from the National Institute of Neurological Disorders and Stroke, USA.

Author information

Authors and Affiliations

Authors

Contributions

L.M. and H.Z. designed the experiments. L.M. and J.D.-C. performed the experiments (with indispensable assistance from O.J.B., M.A.M. and J.B.D.). L.M., J.D.-C. and H.Z. analysed the data. M.Q. bred and maintained the transgenic mice. T.M. and H.Z. supervised the project and secured the funding. L.M. and H.Z. wrote the first draft. All authors critically edited the paper.

Corresponding author

Correspondence to Haining Zhong.

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Extended data figures and tables

Extended Data Fig. 1 dSPNs and iSPNs exhibit cell-specific set points of basal PKA activity.

a, Example projections from dSPNs and iSPNs, as visualized by Cre-dependent GFP expression in the dorsal striatum of Drd1a-cre (5 mice) and Adora2a-cre mice (2 mice), respectively. GPe: globus pallidus, external segment; SNr: substantia nigra pars reticulata; inj: injection site; proj: projection site. b, Representative post hoc histology section of an animal with GRIN lens implanted and tAKARα expressed in iSPNs, and centers of GRIN lens implantations for dSPNs (blue, 17 mice) and iSPNs (magenta, 15 mice), as mapped onto nearby coronal sections from Franklin & Paxinos (2007)57. Section positions in millimeters anterior to bregma are indicated. c, Comparison of basal tAKARα lifetimes between dSPN or iSPN somata and their corresponding dendrites. n (neurons/mice) = 16/4 for dSPN and 22/6 for iSPN. Between somata and dendrites, two-tailed paired Student’s t-test, from left to right, p = 1.4x10−4 and 1.5x10−4; dF = 15 and 21; t = 5.1 and 4.6. Between somata of dSPNs and iSPNs, two-tailed unpaired Student’s t-test, p = 0.79; dF = 36; t = 0.26. d, Collective changes of basal PKA activity responding to isoflurane (isofl.) exposure (1.5%). n (neurons/mice) = 52/5 for dSPNs and 71/7 for iSPNs. Two-tailed paired Student’s t-test, from left to right, p = 5.8x10−25 and 3.5x10−32; dF = 51 and 70; t = −19.2, and −21.2. e & f, Representative intensity and corresponding lifetime (LT) images across two days (e) and correlation of basal lifetimes of the same cells (f) for dSPNs and iSPNs. The p values are from the fit. g, Basal lifetimes of the same cells across seven consecutive days. h, Correlation of basal lifetimes with the average fluorescence intensity of the corresponding cells. The p values are from the fit. For panels eh: n (neurons/fields of view [FOVs]/mice) = 52/12/5 for dSPNs and 86/19/8 for iSPNs. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05; ***: p ≤ 0.001. Brain illustration in b is adapted from ref. 57.

Source data

Extended Data Fig. 2 tAKARα response to pharmacological manipulations depends on sensor phosphorylation.

a & b, Collective responses of tAKARα (snsr) and its phosphorylation-deficient mutant (mut.) to the indicated drug application in dSPNs (a) and iSPNs (b). From left to right, n (neurons/mice) = 38/6, 54/4, 38/6, and 41/4 for dSPNs, and 63/8, 57/4, 63/8, and 60/4 for iSPNs. Two-tailed unpaired Student’s t-test for both panels, from left to right, p = 1.7x10−21, 0.029, 6.0x10−6, and 2.0x10−12; dF = 90, 77, 118, and 121; t = 12.6, 2.2, 4.7, and −7.8. All error bars represent SEM and their centers represent the mean. *: p ≤ 0.05; ***: p ≤ 0.001.

Source data

Extended Data Fig. 3 Supporting data for PKA activity elicited by optogenetic dopamine releases.

a & b, Representative trace (a) and dose-response curve (b) of PKA response in a dSPN elicited by different numbers of trains (1 train/s of 20 Hz 10 x 1.5-ms blue light [470 nm] pulses) of optogenetic stimulation. n (neurons/mice) = 38/5. c, Representative traces of PKA responses to 10 trains of optogenetic stimulation of dopamine release before (top) and after (bottom) intraperitoneal injection of SKF83566 (SKF). Black curve shows a single-exponential fit of the decaying phase of the response. d, The collective τoff of optogenetically-induced PKA responses. To achieve a high signal-to-noise ratio for proper fitting, PKA signals from an entire field of view, which included 3–5 neurons, were integrated. n (FOVs/mice) = 4/3. e, Collective PKA responses to 10 trains of optogenetic stimulation of dopamine release before and after intraperitoneal injection of SKF83566 (SKF). n (neurons/mice) = 14/3. Two-tailed paired Student’s t-test, p = 4.8x10−5; dF = 13; t = −6.0. f, Collective basal PKA activity responses to the D2R agonist, quinpirole (quin; 1 mg/kg). n (neurons/mice) = 43/4. Two-tailed paired Student’s t-test, p = 1.0x10−10; dF = 42; t = −8.5. All error bars represent SEM and their centers represent the mean. ***: p ≤ 0.001.

Source data

Extended Data Fig. 4 Supporting experiments for manipulating PKA activities using drug administration or PKI expression.

a & b, PKA activity responses to Rp-8-Br0cAMPS (Rp) and H89 for dSPNs (a) and iSPNs (b). From left to right, n (neurons/mice) = 27/3 and 22/3 for dSPNs, and 33/4 and 24/4 for iSPNs. Two-tailed paired Student’s t-test, p = 6.9x10−10 and 2.7x10−5, dF = 26 and 21, t = −9.4 and −5.3 for panel a; and p = 4.0x10−6 and 6.3x10−5, dF = 32 and 23, t = −5.5 and −4.9 for panel b. c, Representative images of co-expression of PKI (red) and the PKA sensor tAKARα (green) in the same cells (top), and representative traces of enforced running-induced PKA activity in neurons of the mouse motor cortex without (bottom left) and with (bottom right) PKI expression. n (FOVs/mice) = 19/9 without PKI and 14/4 with PKI. d, Collective enforced running-induced PKA responses in neurons of mice without or with PKI expression. n (neurons/mice) = 124/9 for control and 30/4 for PKI. Boxes indicate 25th and 75th percentile, with black lines indicating median and whiskers indicating 2.7x standard deviation. Two-tailed unpaired Student’s t-test, p = 1.4x10−7; dF = 152; t = 5.5. e, Representative 3D reconstructions of PKI expression in Drd1a-cre (left) and Adora2a-cre (right) mice. Inj.: injection; proj.: projection; A: anterior; L: lateral; and D: dorsal. f & g, Accelerated rotarod training for mice with PKI or a non-functional PKI mutant (PKImut) expressed in dSPNs (f) or iSPNs (g). n (mice) = 11 for both groups for dSPNs and 10 for both groups for iSPNs. Two-tailed paired Student’s t-test, for first and second days, respectively, dF = 10 and 10, t = −3.3 and −3.2 for dSPNs; and dF = 9 and 9, t = −2.6 and −1.5 for iSPNs. h, Averaged travel velocity of mice with PKI or a non-functional PKI mutant (PKImut) expressed in dSPNs or iSPNs freely moving in an open-field box (37 x 37 cm). From left to right, n (neurons/mice) = 11, 11, 10, and 10. Two-tailed paired Student’s t-test, p = 0.03 and 0.18; dF = 10 and 9; t = 1.2 and −0.6. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Source data

Extended Data Fig. 5 Additional data for voluntary running-induced PKA activity.

a, Representative single-cell traces of voluntary running-induced PKA response in a dSPN (top) and iSPN (bottom). b, Representative traces of integrated PKA response within an entire FOV elicited by short-duration (<30 s) voluntary running in a dSPN (top) and iSPN (bottom) overlaid with the fit for on- and off-phases (black). The entire FOV was integrated to achieve higher photon count and thereby signal-to-noise ratio, and the imaging rate was every 3 s. c, The kinetic time constant of short-duration voluntary running-induced PKA activity in dSPNs and iSPNs. n = 9 FOVs for dSPNs and 10 for iSPNs. Two-tailed Student’s t-test, from left to right dF = 17 and 17; t = 0.34 and −0.17. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05.

Source data

Extended Data Fig. 6 Additional data for enforced running-induced PKA activity.

a & b, Example trace (inset) and correlation of PKA responses of dSPNs (a) and iSPNs (b) to two consecutive enforced running trials in SPNs. n (neurons/mice) = 50/5 for dSPNs and 68/8 for iSPNs. The p values are from the fit. c, Representative single-cell traces of PKA response in a dSPN (top) and iSPN (bottom) elicited by different durations of enforced running. d, Running duration–PKA response relationship of dSPNs and iSPNs. n (neurons/mice) = 85/7 for dSPNs and 121/9 for iSPNs. e, Representative traces of PKA responses in a dSPN (top) and iSPN (bottom) elicited by a short duration (25s) of enforced running overlaid with the fits (black) for on- and off-phases. The entire FOV was integrated to achieve higher photon count and thereby signal-to-noise ratio, and the imaging rate was every 3 s. f & g, Comparison of the kinetic time constants of PKA responses between short-duration voluntary running and enforced running in dSPNs (f) and iSPNs (g). From left to right, n = 9, 10, 9, and 10 in panel f; and 10, 11, 10, and 11 in panel g. The voluntary running data are the same as those in Extended Data Fig. 4c. Two-tailed unpaired Student’s t-test, from left to right, p = 0.73, 0.87,0.15, and 0.52; dF = 17, 17, 19, and 19; t = 0.35, −0.17, 1.50, and −0.65. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05.

Source data

Extended Data Fig. 7 A2AR antagonist blocks running-induced PKA activity.

a & b, Collective changes of basal (a) and enforced running-induced PKA activity (b) in iSPNs in response to the indicated intraperitoneal injection of two A2A receptor antagonists, istradefylline (istra.) and SCH58261 (SCH). n (neurons/mice) = 63/8, and 49/4 for SCH58261. Two-tailed paired Student’s t-test, from left to right, p = 3.1x10−16, 2.6x10−11, 1.2x10−10, and 6.9x10−12; dF = 62, 48, 62, and 48; t = −11.0, −8.6, −7.7, and −10.3. c, Representative traces of voluntary running-induced PKA response in an iSPN before and after intraperitoneal injection of istradefylline (istra, 2 mg/kg). d, Collective voluntary running-induced PKA activity in iSPNs before and after istradefylline administration. n (neurons/mice) = 46/4. Two-tailed paired Student’s t-test, p = 1.9x10−7; dF = 45; t = −6.2. e & f, Collective changes of basal (e) and enforced running-induced PKA activity (f) in dSPNs and iSPNs in response to the local infusion of saline (1 µL). n (neurons/mice) = 27/7 for dSPNs and 16/5 for iSPNs. Two-tailed paired Student’s t-test, dF = 26, and 15, t = −0.1 and −1.7 for panel e; dF = 26 and 15 t = −1.3 and 1.4 for panel f. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05; ***: p ≤ 0.001.

Source data

Extended Data Fig. 8 The effect of an A1R antagonist on dSPNs and iSPNs.

a & b, Collective changes of basal and enforced running-induced PKA activity, as indicated, in dSPNs (a) and iSPNs (b) in response to the intraperitoneal injection of the A1 receptor antagonist DPCPX (2 mg/kg). n (neurons/mice) = 55/5 for dSPNs and 118/6 for iSPNs. Two-tailed paired Student’s t-test, from left to right across panels, p = 8.4x10−11, 0.0029, 3.9x10−4, and 0.42; dF = 54, 54, 117, and 117; t = −8.0, −3.1, −3.7, and 0.8. All error bars represent SEM and their centers represent the mean. n.s.: p > 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

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Extended Data Fig. 9 Voluntary running-induced adenosine release.

a, Representative histology section of wildtype mice (n = 9) with an optical fibre implanted and GRABAdo1.0 expressed in neurons (under a synapsin promotor) of the dorsal lateral striatum. b, Example fibre photometric recording traces, aligned to an enforced running bout. c, Average voluntary running-elicited adenosine responses (top) and the corresponding running (bottom) aligned to movement initiations. n (bouts/mice) = 86/5 for GRABAdo1.0 and 34/2 for mutant. d, Average enforced running-induced adenosine release with (black) and without (green) intraperitoneal injection of istradefylline (istra, 2 mg/kg). n (bouts/mice) = 12/6 for both control and istradefylline. e, Collective results of the experiment in panel d. n = 6 mice. Two-tailed paired Student’s t-test, p = 8.6x10−4; dF = 5; t = 7.1. All error bands represent SEM and their centers represent the mean. ***: p ≤ 0.001.

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Supplementary information

Reporting Summary

Supplementary Video 1

Representative PKA activity in dSPNs during voluntary running. Left, time-lapse pseudo-coloured lifetime images of representative tAKARα-expressing dSPNs during voluntary running. Right, representative lifetime trace (top) and the corresponding mouse movement speed (bottom) of the dSPN circled in the images. Video speed is ×40 of real time.

Supplementary Video 2

Representative PKA activity in iSPNs during voluntary running. Left, time-lapse pseudo-coloured lifetime images of representative tAKARα-expressing iSPNs during voluntary running. Right, representative lifetime trace (top) and the corresponding mouse movement speed (bottom) of the iSPN circled in the images. Video speed is ×40 of real time.

Supplementary Video 3

Representative PKA activity in dSPNs during enforced running. Left, time-lapse pseudo-coloured lifetime images of representative tAKARα-expressing dSPNs during enforced running (about 5 cm s–1). Right, representative lifetime trace (top) and the corresponding mouse movement speed (bottom) of the dSPN circled in the images. Video speed is ×40 of real time.

Supplementary Video 4

Representative PKA activity in iSPNs during enforced running. Left, time-lapse pseudo-coloured lifetime images of representative tAKARα-expressing iSPNs during enforced running (about 5 cm s–1). Right, representative lifetime trace (top) and the corresponding mouse movement speed (bottom) of the iSPN circled in the images. Video speed is ×40 of real time.

Supplementary Video 5

Representative calcium activity (GCaMP6s) in dSPNs during enforced running. Example GCaMP6s imaging of dSPNs in the dorsolateral striatum during enforced locomotion (about 5 cm s–1). The resting and running states are indicated. Video speed is ×8.6 of real time.

Supplementary Video 6

Representative calcium activity (GCaMP6s) in iSPNs during enforced running. Example GCaMP6s imaging of iSPNs in the dorsolateral striatum during enforced locomotion (about 5 cm s–1). The resting and running states are indicated. Video speed is ×8.6 of real time.

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Ma, L., Day-Cooney, J., Benavides, O.J. et al. Locomotion activates PKA through dopamine and adenosine in striatal neurons. Nature 611, 762–768 (2022). https://doi.org/10.1038/s41586-022-05407-4

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