The adenosine-A_2AR-PKA axis in iSPNs: a novel contributor to regulation of locomotion

Article summary

In a recent paper published in Nature, Ma et al. [1] unexpected found that the protein kinase A (PKA) activity increased in indirect pathway spiny projection neurons (iSPNs) of the dorsolateral striatum (DLS) during locomotion via in vivo PKA imaging in individual iSPNs. Pharmacological blockade indicated that A_2A adenosine receptors (A_2AR) rather than A₁R contribute to the increase of PKA activity in iSPNs. In addition, increased in vivo adenosine levels in DLS were detected using the adenosine sensor, GRABAdo1.0, and was largely abolished by systemic administration of the A_2AR antagonist, istradefylline. This implied that locomotion-induced accumulation of adenosine from breakdown of extracellular ATP in the striatum is the source of increased PKA activity in iSPNs. Therefore, the adenosine-A_2AR-PKA axis in iSPNs is proposed to be a novel contributor to the fine-tuning of locomotion.

Commentary

Previous studies hypothesised that locomotion is fine-tuned by the balance of released dopamine increasing the activity of PKA in direct pathway spiny projection neurons (dSPNs) by activating D₁ dopamine receptors (D₁R) and decreasing PKA activity in iSPNs by activating D₂ dopamine receptors (D₂R) [2]. However, this hypothesis now must take into consideration the new evidence on the role of adenosine in the increase of PKA activity in iSPNs [1].

In this study, the authors first tested the feasibility of in vivo PKA imaging in the DLS and then recorded dynamic PKA activity in SPNs under basal conditions. Application of this approach together with intraperitoneal injections of dopamine receptor antagonists (D₁R: SKF83566; D₂R: eticlopride) found that tonic release of dopamine increased PKA activity in dSPNs, whereas it suppressed PKA in iSPNs. In addition, phasic release of dopamine via optogenetic stimulation of dopaminergic neurones led to increased PKA activity in dSPNs, which was blocked by the D₁R antagonist, SKF83566. However, decreased PKA activity in iSPNs was only seen following systemic administration of the D₂R agonist, quinpirole, and not by optogenetic stimulation-induced dopamine release in both awake and anaesthetized mice. Thus, the changes of PKA activity are consistent with release of dopamine and stimulation of D₁R in dSPNs and D₂R in iSPNs, respectively.

However, an unexpected result was seen when the team employed in vivo PKA imaging to measure PKA activity in dSPNs and iSPNs during locomotion. An increase rather than the expected decrease in PKA activity was detected in iSPNs although increased PKA activity in dSPNs was confirmed by this dynamic in vivo monitoring technique. The alteration of PKA activity was then identified to be responsible for locomotion using two different antagonists, Rp-8-Br-cAMPS and H89 to disrupt PKA activity. Further experiments using the two dopamine receptor antagonists, SKF83566 and eticlopride, revealed that the increased PKA activity in iSPNs was not driven by dopamine, even though the increased PKA activity in dSPNs depends on dopamine. So the question was, which neuromodulator was responsible and how did it contribute to the enhanced PKA activity in iSPNs during locomotion?

Dopamine-adenosine interactions are recognized as an important link in locomotion, tremor, or movement-related disorders (for instance, Huntington’s and Parkinson’s disease) [2, 3]. Given that the adenosine A_2AR is selectively expressed in iSPNs in the striatum and couple to the Gs pathway [4], activation of the A_2AR was proposed by the authors to underlie the increased PKA activity in iSPNs. First, they applied the A_2AR antagonists, istradefylline, and SCH58261, and found that systemic administration of either resulted in decreased PKA activity in iSPNs under resting conditions. Next, local infusion into the striatum of either istradefylline or another A_2AR antagonist, MSX-3, reduced basal PKA activity and abolished locomotion-induced PKA activity in iSPNs. In contrast, basal PKA activity in dSPNs was not altered and locomotion-induced PKA activity only slightly reduced (26% decrease in dSPNs VS 83% decrease in iSPNs). To rule out the involvement of A₁R, the authors employed the A₁R antagonist, DPCPX, and found that it decreased basal PKA activity while increasing locomotion-elicited PKA activity in dSPNs. In contrast, DPCPX caused a small decrease in iSPN basal PKA activity and had no effect on locomotion-elicited PKA activity in iSPNs. Thus, it is clear that the A_2AR in iSPNs plays a key role in the modulation of locomotion-induced increased PKA activity.

Given the fact that the A_2AR is activated by adenosine, real-time and dynamic changes in extracellular adenosine levels in DLS were then studied using the in vivo adenosine sensor, GRABAdo1.0, which has been established to effectively monitor the dynamic adenosine concentration in the brain [5]. Accumulation of adenosine was detected and it was largely abolished by systemic administration of the A_2AR antagonist, istradefylline. The source of adenosine was confirmed by means of local infusion of APCP to antagonize CD73, which showed that the increased adenosine was in part mediated by the extracellular metabolism of ATP.

Based on these data, it is reasonably to propose that the adenosine-A_2AR-PKA axis in iSPNs plays an essential role in locomotion. To explore how adenosine-induced PKA activity in iSPNs affects locomotion, the team used pharmacological blockade of A_2AR and compared it to the blockade of dopamine receptors. The data showed that systemic injection of A_2AR antagonist, istradyfelline, caused acute increases in animal locomotion on the treadmill, whilst both the D₁R antagonist, SKF83566 and the D₂R antagonist, eticlopride, led to decreased animal locomotion. Simultaneous administration of eticlopride (D₂R antagonist) and istradyfelline (A_2AR antagonist) indicated the effects of these two drugs cancelled each other out, which confirmed the hypothesis that both adenosine and dopamine modulate animal locomotion, but in opposing directions: dopamine works like pushing down on the gas pedal of a car, resulting in movement; adenosine works like applying the brake, slowing movement [https://www.nourianzhcp.com/assets/pdf/NOURIANZConsumerBrochureDigital.pdf].

Finally, the authors applied in vivo two-photon imaging of GCaMP6s via the GRIN lens to determine how A_2AR and dopamine receptor antagonism affect intracellular Ca²⁺ levels in dSPNs and iSPNs during animal locomotion. They found that enforced locomotion induced a large increase in cytoplasmic Ca²⁺ in dSPNs or iSPNs. Blocking D₁R with SKF83566 resulted in a 65% decrease of the change in Ca²⁺ levels in dSPNs compared with a 24% decrease in iSPNs. When the D₂R antagonist, eticlopride, was used, the data indicated that cytoplasmic Ca²⁺ increased in iSPNs, but no detectable change was found in dSPNs. Systemic injection of istradefylline to inhibit A_2AR resulted in a significant reduction (46%) of cytoplasmic Ca²⁺ concentration in iSPNs, but a small increase (30%) in dSPNs, which derived from reduced lateral inhibition from iSPNs.

To summarize, this work provides evidence that the adenosine-A_2AR-PKA axis in iSPNs is a novel contributor to the fine-tuning of locomotion and any component of this axis (adenosine, A_2AR, or PKA) would be a promising target for movement-related disorders derived from striatal dysfunction.

Zhi-Wen Yuan

is currently a master student supervised by Prof. Yong Tang from International Collaborative Centre on Big Science Plan for Purinergic Signalling, Chengdu University of Traditional Chinese Medicine. Her research is focusing on purinergic signaling and acupuncture mechanism.

Authors’ contributions

ZWY and YT wrote the main manuscript text and all authors reviewed the manuscript.

Funding

This work was financial support from the NSFC (82274668, 82230127).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interests

The authors declare that they have no competing interests.