PKA, influenced by retrograde signalling from muscle contraction, enhances neurotransmission at the neuromuscular junction (NMJ). This occurs through the phosphorylation of key molecules in synaptic vesicle exocytosis, SNAP-25 and Synapsin-1. The BDNF/TrkB pathway, augmented by muscle contraction, is a known regulator of retrograde control in ACh exocytosis at the NMJ. This modulation, including muscarinic receptors M1 and M2 (mAChRs) linked with both PKA and BDNF/TrkB signalling, has not been studied until now. Here, we investigate how synaptic activity and muscle contraction regulate PKA subunits through BDNF/TrkB and mAChRs, impacting neurotransmission. To investigate synaptic retrograde regulation on PKA subunits, rat phrenic nerve was stimulated (1Hz, 30min) with or without contraction (abolished by μ-conotoxin GIIIB) and applied pharmacological treatments targeting TrkB, M1 and M2 mAChRs, and exogenous BDNF. Western blotting and IHC were used to detect protein levels and phosphorylation in the diaphragm muscle. Our findings revealed the essential role of the BDNF/TrkB pathway in regulating PKA subunits for accurate control of synaptic vesicle exocytosis. TrkB did not affect PKA catalytic subunits Cα and Cβ levels but regulated PKA RIα and RIIβ to facilitate phosphorylation of key targets (SNAP-25 and Synapsin-1), while muscarinic receptors pathway maintained a balance in this regulatory activity. These findings describe PKA subunit dynamics under TrkB signalling, M1 and M2 mAChRs pathways, and pre- and postsynaptic activity, highlighting specific role of the BDNF/TrkB pathway in retrograde regulation. This contributes to understanding bidirectional communication for precise ACh release, offering potential therapeutic insights for conditions with impaired neuromuscular crosstalk.Funding:2021SGR01214,PID2019-106332GB-I00,PID2022-141252NB-I00,PRE2020-092084,2021-FI-B00755,2023PMF-PIPF-15.