Dominant Role of βγ Subunits of G-Proteins in Oxytocin-Evoked Burst Firing

Abstract

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 pM OT for 30 min or progressively rising OT concentrations from 1 to 100 pM 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-␤-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/oproteins, did not block OT-evoked bursts, suggesting that G i /G o activation is unnecessary for burst generation. Thus, we further examined G␣ q/11 -associated signaling pathways in OT-evoked bursts. Inhibition of phospholipase C or RhoA/Rho kinase did not block bursts. Activation of G␤␥ subunits using myristoylated G␤␥-binding peptide (mSIRK) caused bursts, whereas intracellularly loaded antibody against G␤ 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␤ subunits from G␣ q/11 subunits shown by coimmunoprecipitation and immunocytochemistry, supporting crucial roles for OTRs and G␤␥ subunits in the milk-ejection reflex. We conclude that G␤␥ subunits play a dominant role in burst firing evoked by applied OT or by suckling.

Interaction of Extracellular Signal-Regulated Protein Kinase 1/2 with Actin Cytoskeleton in Supraoptic Oxytocin Neurons and Astrocytes: Role in Burst Firing

“…OT is essential for burst generation by OT neurons in intact animals (Freund-Mercier and Richard, 1982) and in slices (Wang and Hatton, 2007). Similar to our previous results (Wang and Hatton, 2007), bath application of 10 pM OT for 30 min in normal aCSF evoked bursts in 25% (6 of 24) of OT neurons ( Fig.…”

“…After incubation with goat polyclonal OT-NP antibody (1:250 to 1:400 dilution) for 4 h at room temperature, a donkey anti-goat antibody (Alexa Fluor 647 labeled, 1:1000) was applied for 1.5 h to label OT neurons. In separate slice experiments used for identification of other antigens, slices were exposed to drugs only, after incubation at room temperature for 2-4 h as described previously (Wang and Hatton, 2007). Slices were treated with various agents, fixed and permeated, and then incubated with primary antibodies at 4°C overnight (mouse antipERK1/2, 1:1000) or room temperature for 4 h (goat anti-OT-NP, 1:250 to 1:400; rabbit anti-GFAP, 1:400).…”

“…Samples were from either slices or intact animals, as described previously (Wang and Hatton, 2007). Animals were decapitated, and the SONs were dissected out.…”

“…Blocking activation of OT receptors (OTRs) blocked suckling-evoked bursts (Freund-Mercier and Richard, 1984). OT can also evoke bursts in OT neurons in brain slices (Wang and Hatton, 2007) that preserve most features of the in vivo bursts (Wang and Hatton, 2004). These characteristics make OT-evoked in vitro bursts and milk ejections suitable models for studying cellular mechanisms involved in burst generation.…”

“…In the signaling pathway for OT actions, pERK1/2 has been identified (Hoare et al, 1999;Zlatnik et al, 2000;Burns et al, 2001). OTR-associated G-protein ␤␥ subunits, a dominant signal in OT-evoked bursts (Wang and Hatton, 2007), can activate ERK1/2 by phosphorylation to form pERK1/2 (Zhong et al, 2003). Another major target of OT actions is actin.…”