Role of M₄ -receptor cholinergic signaling in direct pathway striatal projection neurons during dopamine depletion

Direct pathway striatal projection neurons (dSPNs) are characterized by the expression of dopamine (DA) class 1 receptors (D₁ R), as well as cholinergic muscarinic M₁ and M₄ receptors (M₁ R, M₄ R). D₁ R enhances neuronal firing through phosphorylation of voltage-gate calcium channels (Ca_V 1 Ca²⁺ channels) activating Gs proteins and protein kinase A (PKA). Concurrently, PKA suppresses phosphatase PP-1 through DARPP-32, thus extending this facilitatory modulation. M₁ R also influences Ca²⁺ channels in SPNs through Gq proteins and protein kinase C. However, the signaling mechanisms of M₄ R in dSPNs are less understood. Two pathways are attributed to M₄ R: an inhibitory one through Gi/o proteins, and a facilitatory one via the cyclin Cdk5. Our study reveals that a previously observed facilitatory modulation via Ca_V 1 Ca²⁺ channels is linked to the Cdk5 pathway in dSPNs. This result could be significant in treating parkinsonism. Therefore, we questioned whether this effect persists post DA-depletion in experimental parkinsonism. Our findings indicate that in such conditions, M₄ R activation leads to a decrease in Ca²⁺ current and an increased M₄ R protein level, contrasting with the control response. Nevertheless, parkinsonian and control actions are inhibited by the Cdk5 inhibitor roscovitine, suggesting Cdk5's role in both conditions. Cdk5 may activate PP-1 via PKA inhibition in DA depletion. Indeed, we found that inhibiting PP-1 restores control M₄ R actions, implying that PP-1 is overly active via M₄ Rs in DA-depleted condition. These insights contribute to understanding how DA-depletion alters modulatory signaling in striatal neurons. Additional working hypotheses are discussed.