Activity-dependent synapse reduction is a major determinant of neuromu
scular innervation. Previous research has shown that nanomolar concent
rations of hirudin, a specific thrombin antagonist, significantly atte
nuates this reduction, and protease nexin 1 (PN1), an endogenous throm
bin inhibitor closely localized to the neuromuscular synapse, can inhi
bit synapse reduction at similar concentrations. Protease inhibitors w
hich do not inhibit thrombin, including cystatin and aprotinin, had no
effect on synapse reduction. We present a series of experiments exami
ning whether prothrombin and/or PN1 gene expression, as well as thromb
in activity, are regulated in muscle cultures by acetylcholine (ACh) r
eceptor activation. We also studied the effect of exogenous thrombin o
n synapse elimination in co-cultures of muscle and cholinergic neurons
. Cultured muscle cells were electrically blocked with tetrodotoxin (T
TX), or co-treated with ACh in order to isolate ACh receptor activatio
n. Electrical blockade resulted in a decrease in thrombin release to a
bout two-thirds of control values. The application of ACh to electrica
lly blocked muscle cultures resulted in a 2.5-fold increase in thrombi
n activity released into the medium and a 2-fold increase in prothromb
in gene expression. In contrast, ACh treatment in the presence of TTX
had no effect on PN1 gene expression compared to treatment with TTX al
one. In addition, exogenous thrombin significantly increased synapse e
limination in unstimulated muscle/cholinergic neuron co-cultures. Thes
e results suggest that thrombin or a thrombin-like molecule released f
rom muscle is required for activity-dependent synapse elimination and
is regulated by neuromuscular activity. (C) 1997 Elsevier Science B.V.