Climbing fiber activity reduces 14-3-3-θ regulated GABAA receptor phosphorylation in cerebellar Purkinje cells

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABAA receptors (GABAARs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. Although long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABAARs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABAAg2, and reduced the interaction of 14-3-3-θ with protein kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABAAγ2. Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABAAγ2. Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABAAγ2 and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell-surface expression of GABAARs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity.

▶Climbing fibers activate signaling proteins that regulate the GABAA receptors in Purkinje cells. ▶Climbing fiber activity decreases 14-3-3-θ and serine phosphorylation of GABAAγ2. ▶Climbing fiber activity also decreases interaction of 14-3-3-θ with PKCγ. ▶Knockdown 14-3-3-θ in vivo reduces serine phosphorylation of GABAAγ2. ▶Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduces serine phosphorylation and cell-surface expression of GABAAγ2.