Cholinergic stimulation increases thrombin activity and gene expression in cultured mouse muscle

Activity-dependent synapse reduction is a major determinant of neuromuscular innervation. Previous research has shown that nanomolar concentrations of hirudin, a specific thrombin antagonist, significantly attenuates this reduction, and protease nexin 1 (PN1), an endogenous thrombin inhibitor closely localized to the neuromuscular synapse, can inhibit synapse reduction at similar concentrations. Protease inhibitors which do not inhibit thrombin, including cystatin and aprotinin, had no effect on synapse reduction. We present a series of experiments examining whether prothrombin and/or PN1 gene expression, as well as thrombin activity, are regulated in muscle cultures by acetylcholine (ACh) receptor activation. We also studied the effect of exogenous thrombin on synapse elimination in co-cultures of muscle and cholinergic neurons. Cultured muscle cells were electrically blocked with tetrodotoxin (TTX), or co-treated with ACh in order to isolate ACh receptor activation. Electrical blockade resulted in a decrease in thrombin release to about two-thirds of control values. The application of ACh to electrically blocked muscle cultures resulted in a 2.5-fold increase in thrombin activity released into the medium and a 2-fold increase in prothrombin gene expression. In contrast, ACh treatment in the presence of TTX had no effect on PN1 gene expression compared to treatment with TTX alone. In addition, exogenous thrombin significantly increased synapse elimination in unstimulated muscle/cholinergic neuron co-cultures. These results suggest that thrombin or a thrombin-like molecule released from muscle is required for activity-dependent synapse elimination and is regulated by neuromuscular activity.