Identification of the key molecules that bridge presynaptic neuronal events and long-term modification of the postsynaptic process is an important challenge which will have to be met in order to further our understanding of the mechanisms for learning and memory. This study is focused on neuregulin-1 (NRG-1), a neurotrophic factor, that is known to regulate the development of various tissues and/or the life/death of cells through activation of the ErbB family receptor tyrosine kinases. It was discovered that the soluble form of NRG-1 (sNRG-1) is produced from the transmembrane form of NRG through proteolytic cleavage during electrical stimulation of either cultured cerebellar granule cells (GCs) or pontine nucleus neurons (PNs) that are presynaptic to GCs. sNRG-1 was assayed by measuring the phosphorylation of both the ErbB receptors and cyclic AMP-responsive element-binding protein (CREB), and by means of antibodies to sNRG-1. The cleavage and release of NRG-1 depended on the frequency of electrical stimulation; the peak effect was at 50 Hz in both GCs and PNs. Activation of protein kinase C (PKC) mimicked this effect. The culture apparatus provided along with the mass-electrical stimulation that was employed proved to be a powerful tool for combining neuronal electrical events and chemical events. We conclude from the results that, in mossy fibre (PN axon)-GC synapses, electrical activity controls the proteolytic processing of NRG-1 in a frequency-dependent fashion and involves PKC. Furthermore, cleaved sNRG-1 plays an important functional role in regulating transmission across these synapses. Keywords: activity-dependent, cyclic AMP-responsive element-binding protein (CREB), ErbB, neuregulin, proteolytic cleavage, synapses and cerebellum. Abbreviations used: contlGCs, GC-conditioned media that were incubated with pre-immune serum IgG before immunization with peptides of the cleavage site; contlPNs, PN-conditioned media that were incubated with pre-immune serum IgG before immunization with peptides of the cleavage site; CREB, cyclic AMP-responsive elementbinding protein; dep1GCs, GC-conditioned media that were incubated with antisNRG-1b1; dep1PNs, PN-conditioned media that were incubated with antisNRG-1b1; DGCs, GCs transfected with the deleted mNRG; E18, embryonic day 18; GABA A , gamma-aminobutyric acid type A, GC(s), granule cell(s); H7, dihydrochloride; MF(s), mossy fibre(s); mNRG, transmembrane form of NRG; nGCs, non-transfected nGCs; NMDA, N-methyl-D-aspartic acid; nPNs, non-transfected PNs; P7, post-natal day 7; pCREB, phosphorylated CREB; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PN(s), pontine nucleus neuron(s); PSD-95, postsynaptic density-95; rNRG, recombinant NRG1b1 overexpressed and purified by means of an Escherichia coli expression system; sNRG, soluble form of NRG; tGCs, mNRG-transfected GCs; t(K 212
