Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain
Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain
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DOI：
10.1016/s0306-4522(98)00520-x
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发表时间：
1999-01-01
期刊：
影响因子：
3.3
通讯作者：
Nichols, RA
中科院分区：
文献类型：
--
作者：
Nayak, SV;Rondé, P;Nichols, RA
血清素5-羟色胺-3受体是广泛分布于神经系统的配体门控离子通道。在CNS内，大部分5-羟色胺-3受体似乎存在于突触前神经末梢上，并且使用成像方法，先前显示单个分离的神经末梢上的突触前5-羟色胺-3受体来自大鼠纹状体的突触体（突触体）显示出一组独特的性质--慢启动，与位于神经元细胞体突触后位点的5-羟色胺-3受体相比，Ca 2 +-的脱敏作用很小，表观渗透性很高。为了考虑它们的特征性性质是否是突触前5-羟色胺-3受体在整个大脑中的共同特征，我们使用共聚焦显微镜来测量由5-羟色胺3激动剂诱导的突触体中来自代表性大鼠脑区域的反应引起的细胞内Ca 2+浓度的变化，5-羟色胺-3受体的总体表达水平从相对低（小脑）到中等（纹状体和海马）再到高（杏仁核）。应用100 nM间氯苯双胍（一种特异性5-羟色胺-3受体激动剂）可诱导纹状体（约占总浓度的6%）、海马（约占总浓度的3%）、杏仁核（约占总浓度的30%）和小脑（约占总浓度的32%）突触体亚群中相对细胞内Ca2+浓度发生变化。为了确保对5-羟色胺-3激动剂刺激没有反应的突触体的活力，随后加入KCl（45 mM）以使相同的突触体群体去极化，然后在来自所有四个区域的80 - 90%的突触体中观察到细胞内Ca 2+浓度的增加。由K+诱发的去极化产生的突触体内Ca 2+变化的动力学在所有区域都是相似的，显示出快速上升到峰值，随后是明显的平台期。与此相反，由间氯苯双胍引起的细胞内钙浓度的变化显示出明显较慢的动力学，类似于以前的研究结果，但不同的响应突触体从一个区域到另一个。特别是，在氯苯基双胍诱导的变化显着较慢的突触体从杏仁核（上升时间常数，tau = 25秒），当从其他区域（纹状体，tau = 12秒;海马，tau = 9.6秒;小脑，tau = 7秒）的突触体的反应相比。为了使用分子方法独立地证明5-羟色胺-3受体在各个区域的神经末梢上的存在，我们分别使用针对5-羟色胺-3受体的N-末端肽的多克隆抗体和单克隆抗突触体蛋白抗体，对突触体进行5-羟色胺-3受体和突触囊泡蛋白突触体蛋白的双重免疫染色，并观察到5-羟色胺-3受体在不同亚群的突触体从每个区域，提供了直接支持的结果在我们的功能实验。这些结果表明，突触前5-羟色胺-3受体的独特性质被发现在整个大脑，在所研究的脑区域中观察到对激动剂刺激的反应动力学的明显差异。正如预期的那样，对5-羟色胺-3激动剂的应用作出反应的突触体群体的比例在制剂中从一个区域到另一个区域各不相同;然而，与先前的结合研究相比，小脑中突触前5-羟色胺-3受体的比例相对较高，表明该区域中5-羟色胺-3受体的总体密度相对较低。我们假设突触前5-羟色胺-3受体存在于神经末梢上，调节整个大鼠大脑中选择性突触的功能。(C)1999年IBRO。出版社：Elsevier Science Ltd
The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a significant portion of the 5-hydroxytryptamine-3 receptors appears to be present on presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual isolated nerve terminals (synaptosomes) from rat corpus striatum display a distinctive set of properties-slow onset, little desensitization and high apparent permeability for Ca2+-when compared to those observed for 5-hydroxytryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodies. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confocal microscopy to measure changes in intracellular Ca2+ concentration resulting from 5-hydroxytryptamine 3 agonist-induced responses in synaptosomes from representative rat brain regions, ranging in expression of overall levels of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to intermediate (corpus striatum and hippocampus) to high (amygdala). Application of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 receptor agonist, induced changes in relative intracellular Ca2+ concentration in subsets of synaptosomes from the corpus striatum (similar to 6% of total), hippocampus (similar to 3% of total), amygdala (similar to 30% of total) and cerebellum (similar to 32% of total). In order to assure the viability of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCI (45 mM) was subsequently added to depolarize the same population of synaptosomes, and increases in intracellular Ca2+ concentration were then seen in 80-90% of the synaptosomes from all four regions. The kinetics of the intra synaptosomal Ca2+ changes produced by K+-evoked depolarization were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca concentration evoked by m-chlorophenyl biguanide displayed substantially slower kinetics, similar to previous findings, but which varied among responding synaptosomes from one region to another. In particular, in-chlorophenyl biguanide-induced changes were notably slower in synaptosomes from the amygdala (rise time constant, tau = 25 s), when compared to responses in synaptosomes from other regions (striatum, tau = 12 s; hippocampus, tau = 9.6 s; cerebellum, tau = 7 s). To independently demonstrate the presence of 5-hydroxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-hydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal peptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptophysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subsets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments.These results suggest that the distinctive properties of presynaptic 5-hydroxytryptamine-3 receptors are found throughout the brain, with evident differences in the kinetics of the responses to agonist stimulation observed across the brain regions studied. As expected, the proportion of the synaptosomal population that responded on application of 5-hydroxytryptamine-3 agonist varied in preparations from one region to another; however, the presence of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 receptors in the cerebellum contrasts with previous binding studies demonstrating a relatively low overall density of 5-hydroxytryptamine-3 receptors in this region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the functioning of select synapses throughout the rat brain. (C) 1999 IBRO. Published by Elsevier Science Ltd.