Milk Ejection and Its Control

Neuronal firing patterns determine the manner of neurosecretion, the underlying mechanisms of which are poorly understood. Using supraoptic nuclei in brain slices from lactating rats, we examined the involvement of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and filamentous actin (F-actin) in burst generation by oxytocin (OT) neurons. Blocking phosphorylation of ERK1/2 (pERK1/2) decreased miniature EPSCs and blocked OT-evoked bursts, as did intracellularly loading an antibody against pERK1/2. OT (10 pM) increased cytosolic pERK1/2 close to the cell membrane within the first 5 min, subsiding by 30 min, whereas OT elicited pERK1/2 nuclear translocation in closely associated supraoptic astrocytes. The increased pERK1/2 was tightly correlated with spatiotemporal actin dynamics. In OT neurons, OT initially increased F-actin, particularly at membrane subcortical areas, and then decreased it after 30 min. Both polymerization and depolymerization of actin cytoskeleton were associated with bursts, but only polymerization facilitated OTevoked bursts. Blocking ERK1/2 activation blocked OT-evoked actin polymerization, whereas depolymerizing F-actin increased pERK1/2 expression. These changes were further identified in vivo. In intact animals, suckling increased ERK1/2 activation in the cytosol and membrane subcortical area F-actin formation in OT neurons, whereas it increased F-actin concentration in astrocytic somata. Coimmunoprecipitation showed that suckling increased molecular interactions between pERK1/2 and actin. Finally, two different blockers of ERK1/2 kinase injected intracerebroventricularly reduced suckling-evoked milk ejections. This is the first demonstration that OT mediation of suckling-evoked bursts/milk ejections is via interactions between pERK1/2 and actin cytoskeleton.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wang¹,

Hatton²

2007

“…OT is a critical mediator of suckling-evoked MER (Wakerley et al, 1994) and can cause acute retraction of astrocytic processes (Wang and Hatton, 2007b). Thus, it is likely that OT also mediates suckling-elicited GFAP reduction.…”

Section: Suckling-reduced Gfap By Direct Action Of Ot On Astrocytesmentioning

confidence: 99%

Astrocytic Plasticity and Patterned Oxytocin Neuronal Activity: Dynamic Interactions

Wang¹,

Hatton²

2009

123

Astroglial-neuronal interactions are important in brain functions. However, roles of glial fibrillary acidic protein (GFAP) in this interaction remain unclear in acute physiological processes. We explored this issue using the supraoptic nucleus (SON) in lactating rats. At first, we identified the essential role of astrocytes in the milk-ejection reflex (MER) by disabling astrocytic functions via intracerebroventricular application of L-aminoadipic acid (L-AAA). L-AAA blocked the MER and reduced GFAP levels in the SON. In brain slices, L-AAA suppressed oxytocin (OT) neuronal activity and EPSCs. Suckling reduced GFAP in immunocytochemical images and in Western blots, reductions that were partially reversed after the MER. OT, the dominant hormone mediating the MER, reduced GFAP expression in brain slices. Tetanus toxin suppressed EPSCs but did not influence OT-reduced GFAP. Protease inhibitors did not influence OT-reduced GFAP images but blocked the degradation of GFAP molecules. In the presence of OT, transient 12 mM K ϩ exposure, simulating effects of synchronized bursts before the MER, reversed OT-reduced GFAP expression. Consistently, suckling first reduced and then increased the expression of aquaporin 4, astrocytic water channels coupled to K ϩ channels. Moreover, GFAP molecules were associated with astrocytic proteins, including aquaporin 4, actin, and glutamine synthetase and serine racemase. GFAP-aquaporin 4 association decreased during initial suckling and increased after the MER, whereas opposite changes occurred between GFAP and actin. MER also decreased the association between GFAP and glutamine synthetase. These results indicate that suckling elicits dynamic glial neuronal interactions in the SON; GFAP plasticity dynamically reflects OT neuronal activity.

“…Excitatory effects of OT have been explored extensively [for review, see Crowley and Armstrong (1992), Wakerley et al (1994), and Leng et al (1999)], but this is the first identification of the mechanisms underlying the burst-evoking potency of OT in lactating animals. In contrast to most of the previous work, the present study and a few others used effective OT concentrations (Kow et al, 1991;Kuriyama et al, 1993; equal to or lower than the reported physiological level (Neumann et al, 1993).…”

Section: Ot-evoked Excitation and Burstsmentioning

confidence: 99%

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

Wang

Hatton²

2007

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.