【作者】 柯江滨；

【导师】 杨雄里； 钟咏梅； 王中峰；

【作者基本信息】 复旦大学 ， 神经生物学， 2008， 博士

【摘要】 生长抑素（somatostatin,SRIF）是中枢神经系统（central nervous system,CNS）中的一种神经活性肽,通过激活5种特异的受体亚型(sst_1-5)行使其功能,可作为递质或调质起作用。在视网膜中,SRIF主要位于无长突细胞（amacrine cell,AC）上。已有文献表明,sst_1-4在视网膜中均有表达,但在视网膜中仅有较低水平的sst₅mRNA表达。在本工作中,我们研究了sst₅在大鼠视网膜AC上的表达。此外,由于缺少特异性受体拮抗剂（除sst₂外）,有关不同SRIF受体亚型在神经元上生理功能的研究报道还很少。近年来,随着特异性sst₅拮抗剂的发现,为我们研究sst₅在视网膜中的功能提供了可能性。在本工作中,我们首次报道了生长抑素受体5亚型（sst₅）在大鼠视网膜AC上的表达,并进一步研究了SRIF对培养的大鼠视网膜GABA能AC突触前抑制性递质释放的调节。应用免疫细胞化学方法,我们首先研究了sst₅在大鼠视网膜AC上的表达。sst₅的免疫标记弥散地遍布整个内网状层,并在内网状层远端形成两条明显的荧光亮带。免疫双标实验显示,sst₅表达在GABA能AC上。进一步的双标实验显示,sst₅在酪氨酸羟化酶（tyrosine hydroxylase,TH）标记的多巴胺能AC和胆碱乙酰基转移酶（choline acetyl-transferase,CHAT）标记的胆碱能AC上均有表达。免疫阳性标记主要出现在这些细胞的细胞膜和胞体-突起连接部。在胆碱能AC的位于内网状层的突起上也能观察到很弱的sst₅标记。相反,在被Ca²⁺结合蛋白parvalbumin（PV）标记的甘氨酸能AⅡ型AC上并没有sst₅的阳性标记。此外,SRIF与sst₅在多巴胺能和胆碱能AC上共表达。这些结果提示,sst₅在视网膜AC上可能作为自身受体和/或常规受体起作用。应用全细胞膜片钳记录技术,我们进一步研究了SRIF对培养的大鼠视网膜GABA能AC突触前活动的调节。结果发现,在培养GABA能AC上可记录到微小抑制性突触后电流（miniature inhibitory postsynaptic current,mIPSC）,该电流可被GABA_A受体特异性拮抗剂bicuculline（10/μM）完全抑制,说明mIPSC是由突触后GABA_A受体介导的。在此基础上,我们观察到1μM SRIF明显压抑mIPSC的频率,该压抑作用可被sst₅和sst₂的特异拮抗剂部分阻断,说明SRIF是通过激活突触前sst₅和/或sst₂起作用。尽管SRIF略微压抑mIPSC的平均幅度,但并不改变mIPSC的动力学特性,如上升时间（rise time）和衰减时间（decay time）。提高胞外Ca²⁺浓度增加mIPSC的频率,而在胞外高钙条件下,mIPSC频率同样可被1μM SRIF明显压抑。在去除胞外Ca²⁺的情况下,mIPSC几乎被完全压抑,说明mIPSC对胞外Ca²⁺有依赖性。进一步研究发现,200μM CdCl₂阻断绝大部分mIPSC,提示电压门控Ca²⁺通道在其中起重要作用,因此,我们研究了L型Ca²⁺通道阻断剂nimodipine的作用,发现10μM nimodipine明显压抑mIPSC,提示突触前L型Ca²⁺通道与mIPSC密切相关。此外,5μM forskolin（腺苷酸环化酶激活物）增加mIPSC的频率,而PKA的特异性抑制剂Rp-cAMP（20μM）降低mIPSC的频率。在Rp-cAMP存在的情况下,SRIF对mIPSC不再有压抑作用。这些结果提示,SRIF激活特异性受体后可能抑制cAMP-PKA通路,继而限制胞外Ca²⁺通过电压门控Ca²⁺通道的内流,从而减少GABA能AC突触前GABA的释放。综上所述,本工作研究了sst₅在大鼠视网膜AC上的表达,并进一步显示sst₅和/或sst₂部分介导了SRIF对GABA能AC突触前GABA释放的压抑作用,进而探讨了这种压抑作用的可能机制。更多还原

【Abstract】 Somatostatin（SRIF）,a neuroactive peptide in the central nervous system（CNS）, exerts its actions via five subtypes of specific receptors（sst_s） and can work as a neurotransmitter or neuromodulator.In the retina,SRIF is mainly localized to amacrine cells（ACs）.Localization of sst_1-4 in the retina has been immunocytochemically examined in a variety of species,but immunocytochemical data on the expression of sst₅ in the retina are scant.This could be partly because of the rather low sst₅ mRNA levels detected in retinas of several species.In addition, because of lack of specific antagonists（except for sst₂）,there are still limited data on the functional roles of sst_s in neurons.The emergence of commercial available specific sst₅ antagonist makes the study of functions of sst₅ in the retina possible.In this work,the localization of sst₅ was studied immunocytochemically in rat retinal ACs.Labeling for sst₅ was diffusely distributed throughout the full thickness of the inner plexiform layer（IPL） and formed two distinct fluorescence bands in the distal part of the IPL.Double labeling experiments showed that sst₅ was expressed in GABAergic ACs.It was further shown that labeling for sst₅ was observed in both dopaminergic and cholinergic ACs,stained by tyrosine hydroxylase（TH） and choline acetyltransferase（ChAT）,respectively.The immunostaining appeared mainly on the cell membranes and somatodendritic compartments of these ACs.For the cholinergic ACs,weak sst₅-immunoreactivity was also observed in the processes terminating in the IPL.In contrast,no sst₅-immunoreactivity was found in glycinergic AII ACs, stained by parvalbumin（PV）.Furthermore.labeling for SRIF was co-localized with sst₅ in both dopaminergic and cholinergic ACs.These results suggest that sst₅ may serve as an autoreceptor or conventional receptor in retinal ACs.Using whole cell patch-clamp technique,we investigated presynaptic modulation by SRIF in cultured rat retinal GABAergic ACs.Miniature inhibitory postsynaptic currents（mIPSCs） were recorded in cultured rat GABAergic ACs and could be completely inhibited by the GABA_A receptor antagonist bicuculline,indicating that mIPSCs were mediated by postsynaptic GABA_A receptors.1μM SRIF reduced the frequency of mIPSCs,and the effect could be in part blocked by co-application of the sst₅ antagonist BIM 23056 and the sst₂ antagonist CYN-154806.SRIF of 1μM slightly reduced the mean amplitude of mIPSCs,but did not affect the mean rise time and decay time.In addition,the frequency of mIPSCs enhanced by elevating extracellular Ca²⁺ concentration([Ca²⁺]_o) was also reduced by 1μM SRIF,and almost all mIPSC events disappeared after eliminating[Ca²⁺]_o,revealing the[Ca²⁺]_o dependance of mIPSCs.Similarly,200μM CdCl₂ blocked mIPSCs,suggesting that voltage-gated calcium channels（VGCCs） may play an important role in mIPSCs. Indeed,10μM nimodipine,a specific L-type calcium channel blocker,obviously suppressed mIPSCs,suggesting the involvement of L-type calcium channels. Furthermore,forskolin（5μM）,an activator of adenylyl cyclase,enhanced the frequency of mIPSCs,while the PKA inhibitor Rp-cAMP reduced the frequency of mIPSCs.In the presence of Rp-cAMP SRIF had no effect on mIPSCs.These results suggest that SRIF inhibits the Ca²⁺ influx through VGCCs by activating specific receptors,and thus reduces the presynaptic release of GABA from rat retinal ACs via the cAMP-PKA pathway.In conclusion,in the present work,the expression and functions of sst₅ in the rat retinal ACs are systematically studied using immunocytochemical and whole cell patch-clamp techniques.Almost all GABAergic ACs express sst₅.SRIF may depress the presynaptic GABA release from GABAergic ACs via sst₅ and/or sst₂.更多还原