Dominant role of βγ subunits of G-proteins in oxytocin-evoked burst firing

Pulsatile neuropeptide secretion is associated with burst firing patterns; however, intracellular signaling cascades leading to bursts remain unclear. We explored mechanisms underlying burst firing in oxytocin (OT) neurons in the supraoptic nucleus in brain slices from lactating rats. Application of 10 p(M) OT for 30 min or progressively rising OT concentrations from 1 to 100 p(M) induced burst firing in OT neurons in patch- clamp recordings. Burst generation was blocked by OT antagonist and ionotropic glutamate receptor blockers or tetanus toxin. Blocking G- protein activation with suramin or intracellular GDP-beta-S, but not intracellularly administered antibody against the OT-receptor (OTR) C terminus, blocked bursts. Moreover, pretreatment of slices with pertussis toxin, an inhibitor of G(i/o)-proteins, did not block OT-evoked bursts, suggesting that G(i/)G(o) activation is unnecessary for burst generation. Thus, we further examined G alpha(q/11)-associated signaling pathways in OT-evoked bursts. Inhibition of phospholipase C or RhoA/ Rho kinase did not block bursts. Activation of G beta gamma subunits using myristoylated G beta gamma-binding peptide (mSIRK) caused bursts, whereas intracellularly loaded antibody against G beta subunit blocked OT-evoked bursts. Blocking Src family kinase, but not phosphatidylinositol 3-kinase, occluded OT-evoked bursts. Similar to the effects of OT on EPSCs, mSIRK inhibited tonic EPSCs and elicited EPSC clustering. Finally, suckling caused dissociation of OTRs and G beta subunits from G alpha(q/11) subunits shown by coimmunoprecipitation and immunocytochemistry, supporting crucial roles for OTRs and G beta gamma subunits in the milk- ejection reflex. We conclude that G beta gamma subunits play a dominant role in burst firing evoked by applied OT or by suckling.