GABA Activity Mediating Cytosolic Ca2+Rises in Developing Neurons Is Modulated by cAMP-Dependent Signal Transduction

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Abstract
In the majority of developing neurons, GABA can exert depolarizing actions, thereby raising neuronal Ca2+. Ca2+elevations can have broad consequences during development, inducing gene expression, altering neurite outgrowth and growth cone turning, activating enzyme pathways, and influencing neuronal survival. We used fura-2 and fluo-3 Ca2+digital imaging to assess the effects of inhibiting or activating the cAMP signal transduction pathway on GABA activity mediating Ca2+rises during the early stages ofin vitrohypothalamic neural development. Our experiments stemmed from the finding that stimulation of transmitter receptors shown to either activate or inhibit adenylyl cyclase activity caused a rapid decrease in Ca2+rises mediated by synaptically released GABA.Both the adenylyl cyclase activator forskolin and the inhibitor SQ-22,536 reduced the Ca2+rise elicited by the synaptic release of GABA. Bath application of the membrane-permeable cAMP analogs 8-bromo-cAMP (8-Br-cAMP) or 8-(4-chlorophenylthio)-cAMP (0.2–5 mm) produced a rapid, reversible, dose-dependent inhibition of Ca2+rises triggered by synaptic GABA release. Potentiation of GABAergic activity mediating Ca2+rises was observed in some neurons at relatively low concentrations of the membrane-permeable cAMP analogs (20–50 μm). In the presence of tetrodotoxin (TTX), postsynaptic Ca2+rises triggered by the bath application of GABA were only moderately depressed (13%) by 8-Br-cAMP (1 mm), suggesting that the inhibitory effects of 8-Br-cAMP were largely the result of a presynaptic mechanism.The protein kinase A (PKA) inhibitors H89 and Rp-3′,5′-cyclic monophosphothioate triethylamine also caused a large reduction (>70%) in Ca2+rises triggered by synaptic GABA release. Unlike the short-term depression elicited by activation of the cAMP signal transduction pathway, Ca2+depression elicited by PKA inhibition persisted for an extended period (>30 min) after PKA inhibitor washout. Postsynaptic depression of GABA-evoked Ca2+rises triggered by H89 (in the presence of TTX) recovered rapidly, suggesting that the extended depression observed during synaptic GABA release was largely through a presynaptic mechanism. Long-term Ca2+modulation by cAMP-regulating hypothalamic peptides may be mediated through a parallel mechanism.Together, these results suggest that GABAergic activity mediating Ca2+rises is dependent on ongoing PKA activity that is maintained within a narrow zone for GABA to elicit a maximal Ca2+elevation. Thus, neuromodulator-mediated changes in the cAMP-dependent signal transduction pathway (activation or inhibition) could lead to a substantial decrease in GABA-mediated Ca2+rises during early development.