GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABA(A) receptor activation, calcium influx and CaMKII and erk1/2 pathways.

During neuronal development, GABAA-mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1-1000 microm) induces an increase in the intracellular calcium concentration through the activation of GABA(A) receptors and voltage-gated calcium channels of the L-subtype. Perforated patch-clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to - 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium-dependent mechanism triggered by voltage-gated calcium channel activation. Specific blockers of calcium-calmodulin kinase II and mitogen-activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium-dependent kinases.

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.