Obrietan K, van den Pol A N
Department of Biological Sciences, Stanford University, Stanford, California 94305, USA.
J Neurosci. 1997 Jun 15;17(12):4785-99. doi: 10.1523/JNEUROSCI.17-12-04785.1997.
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 of in vitro hypothalamic 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 microM). 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.
在大多数正在发育的神经元中，γ-氨基丁酸（GABA）可发挥去极化作用，从而提高神经元内的钙离子（Ca2+）浓度。在发育过程中，Ca2+浓度升高会产生广泛的影响，包括诱导基因表达、改变神经突生长和生长锥转向、激活酶途径以及影响神经元存活。我们使用fura-2和fluo-3 Ca2+数字成像技术，来评估在体外下丘脑神经发育早期，抑制或激活环磷酸腺苷（cAMP）信号转导途径对介导Ca2+浓度升高的GABA活性的影响。我们的实验源于以下发现：刺激已显示可激活或抑制腺苷酸环化酶活性的递质受体，会导致由突触释放的GABA介导的Ca2+浓度升高迅速降低。腺苷酸环化酶激活剂福斯高林和抑制剂SQ-22,536都能降低由突触释放GABA引起的Ca2+浓度升高。浴槽中加入膜通透性cAMP类似物8-溴-cAMP（8-Br-cAMP）或8-（4-氯苯硫基）-cAMP（0.2 - 5 mM），会对突触GABA释放引发的Ca2+浓度升高产生快速、可逆且剂量依赖性的抑制作用。在一些神经元中观察到相对低浓度（20 - 50 microM）的膜通透性cAMP类似物可增强介导Ca2+浓度升高的GABA能活性。在存在河豚毒素（TTX）的情况下，浴槽中加入GABA引发的突触后Ca2+浓度升高仅被1 mM的8-Br-cAMP适度抑制（13%），这表明8-Br-cAMP的抑制作用主要是由突触前机制导致的。蛋白激酶A（PKA）抑制剂H89和Rp-腺苷3',5'-环磷酸硫代三乙胺也会使突触释放GABA引发的Ca2+浓度升高大幅降低（>70%）。与激活cAMP信号转导途径引起的短期抑制不同，PKA抑制引起的Ca2+浓度降低在PKA抑制剂洗脱后仍持续较长时间（>30分钟）。H89（在存在TTX的情况下）引发的突触后GABA诱发的Ca2+浓度升高的抑制作用迅速恢复，这表明在突触GABA释放过程中观察到的长时间抑制主要是通过突触前机制实现的。cAMP调节下丘脑肽对Ca2+的长期调节可能通过类似机制介导。总之，这些结果表明，介导Ca2+浓度升高的GABA能活性依赖于持续的PKA活性，该活性维持在一个狭窄区域内，以使GABA引发最大的Ca2+浓度升高。因此，神经调质介导的cAMP依赖性信号转导途径的变化（激活或抑制）可能导致早期发育过程中GABA介导的Ca2+浓度升高大幅降低。
