Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain.
Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain.
复制标题
DOI：
10.1186/1472-6750-9-23
复制
发表时间：
2009-03-19
影响因子：
3.5
通讯作者：
Kasparov S
中科院分区：
文献类型：
--
作者：
Benzekhroufa K;Liu B;Tang F;Teschemacher AG;Kasparov S
5-羟色胺（5-HT，serotonin）是哺乳动物脑中的关键神经调质之一，但肾上腺素能神经元的许多基本性质和5-HT释放仍不清楚。本研究的目的是构建一个选择性靶向5-HT能神经元的腺病毒载体系统，并将其应用于大鼠脑内5-HT释放的量子特性研究。我们已经产生了腺病毒载体，其中包含大鼠色氨酸羟化酶-2（TPH-2）基因的3.6 kb片段，该基因选择性（97%与TPH-2共定位）靶向中缝核神经元。为了提高表达水平，采用两步转录扩增策略。这允许通过EGFP荧光直接可视化多巴胺能神经元。使用这些载体，我们已经进行了初步表征的EGFP表达在大鼠器官型脑切片培养的肾上腺素能神经元。使用膜片钳和共聚焦Ca 2+成像鉴定和研究荧光多巴胺能神经元，其特征与先前使用事后鉴定方法报道的特征一致。在未固定的组织中也可以观察到轴突能神经元的精细过程，形态学分析表明两种假定类型的轴突静脉曲张。我们使用微电流分析5 HT释放的量子特性，发现中央5 HT胞吐主要发生在量子的~28000分子从静脉曲张和~34000分子从细胞体。此外，在胞体中，我们观察到少数大的释放事件，平均约80万个分子放电。第一次在哺乳动物大脑中的体细胞-树突细胞室和轴突静脉曲张的5-HT的量子释放已被直接证明和表征。从体树和轴突室释放可能有不同的生理功能。本研究中产生的新型载体开辟了许多新的实验机会，并将大大促进对中枢神经系统的进一步研究。
5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain. We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2) gene which selectively (97% co-localisation with TPH-2) target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules. For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this study open a host of new experimental opportunities and will greatly facilitate further studies of the central serotonergic system.
登录
查看更多内容
影响因子：
56.9
作者：
Santarelli, L;Saxe, M;Hen, R
通讯作者：
Hen, R
影响因子：
16.2
作者：
Bruns, D;Riedel, D;Jahn, R
通讯作者：
Jahn, R
中枢血清素缺乏小鼠的情境恐惧记忆增强
DOI：
10.1073/pnas.0801329105
发表时间：
2008-08-19
影响因子：
11.1
作者：
Dai, Jin-Xia;Han, Hui-Li;Xu, Lin
通讯作者：
Xu, Lin
影响因子：
64.8
作者：
BRUNS, D;JAHN, R
通讯作者：
JAHN, R
影响因子：
5.1
作者：
Nettelbeck, DM;Jérôme, V;Müller, R
通讯作者：
Müller, R