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Neuroscienze

视网膜原酸和皮质化突触功能的电生理学研究

Published: August 07, 2019
doi:
10.3791/59680
, , ,
1Institute of Pathophysiology,University Medical Center of the Johannes Gutenberg University Mainz

Summary

在这里,我们提出了制备含有侧生化核的急性脑切片和视网膜原性和皮质化突触功能的电生理研究方案。该协议提供了一种有效的方法,研究突触与高释放和低释放概率在同一急性脑切片。

Abstract

横向原核是视觉信息的第一个中继站。这个thathamic细胞核的中继神经元整合来自视网膜神经结细胞的输入,并将其投影到视觉皮层。此外,中继神经元从皮层接收自上而下的激发。中继神经元的两个主要兴奋输入在几个方面有所不同。每个中继神经元只接收来自几个视网膜突变突触的输入,这些突触是具有许多释放站点的大型终端。这反映在相对强的激发,中继神经元接收,从视网膜神经结细胞。相比之下,皮质原发性突触更简单,释放位点少,突触强度较弱。两个突触在突触短期可塑性上也有所不同。视网膜突触具有较高的释放概率,因此显示短期抑郁症。相比之下,皮质原化突触的释放概率较低。皮质原性纤维在进入侧生核之前穿过视网膜的丘骨核。视网膜丘脑核(从侧侧原质核旋转)和视道(从侧侧原性核到肠外)的不同位置允许分别刺激皮质原化或视网膜突变突触细胞外刺激电极。这使得侧生核成为理想的大脑区域,可以同时研究两个具有非常不同特性的兴奋突触,以冲击同一细胞类型。在这里,我们描述了一种研究从中继神经元的记录,并执行急性脑切片视网膜原酸和皮质化突触功能的详细分析的方法。本文包含一个分步方案,用于生成侧生核的急性脑切片,以及通过分别刺激视道和皮质化纤维来记录继电器神经元活动的步骤。

Introduction

侧源核的中继神经元整合视觉信息并将其中继到视觉皮层。这些神经元通过视网膜原性突触从神经质化突触接收来自神经细胞的兴奋输入,为中继神经元提供主要的兴奋驱动。此外,中继神经元通过皮质原性突触接收来自皮质神经元的兴奋输入。此外,中继神经元接收来自局部内神经元和GABAergic神经元的细胞内膜1的抑制输入。核视网膜膜像丘拉他和皮层之间的盾牌,使纤维从皮层投射到丘拉他,在相反的方向必须通过核神经膜膜2。

视网膜基质化输入和皮质原产输入显示明显的突触特性3,4,5,6,7,8。视网膜源形成大型终端,具有多个释放站点9、10 。相比之下,皮质基化输入显示具有单释放站点7的小终端。此外,视网膜突触有效驱动继电器神经元的作用电位,尽管仅构成中继神经元3、8、11上所有突触的5-10%。另一方面,皮质原体突触通过控制继电器神经元12、13的膜电位,作为视网膜生成性传输的调节器。

中继神经元的这两个主要兴奋输入在功能上也不同。一个突出的区别是视网膜原性突触的短期抑郁症和皮质原性突触的短期促进3,5,8。短期可塑性是指突触强度在几毫秒到几秒钟的时间段内反复活跃时,突触强度变化的现象。突触释放概率是短期可塑性的重要因素。突触,初始释放概率较低,显示短期便利,由于Ca2+在预突子的积累,因此在重复活动时观察到释放概率的增加。相反,具有高释放概率的突触通常表现为短期抑郁症,因为现成的可释放囊泡的耗竭14。此外,脱敏后受体的脱敏有助于在一些高释放概率突触8,15的短期可塑性。β-氨基-3-羟基-5-甲基-4-异丙酸(AMPA)受体的高释放概率和脱敏性导致视网膜原性突触的突出短期抑郁。相反,低释放概率是皮质原性突触短期促进的基础。

在小鼠中,视道从牛侧位进入背侧原质核(dLGN),而皮质基化纤维进入dLGN。两个输入之间的距离允许调查两个完全不同的兴奋输入冲击到同一细胞的单个属性。在这里,我们建立和改进了前面描述的解剖方法,其中视网膜和皮质化纤维被保存在急性脑切片3中。然后,我们描述了继电器神经元的电生理学研究,以及用细胞外刺激电极刺激视网膜和皮质化纤维。最后,我们提供了一个方案,用于用生物细胞蛋白填充继电器神经元,并随后进行解剖分析。

Protocol

所有实验均获得莱茵兰-普法尔茨地区动物实验政府监督小组的批准。 1. 解决方案 解剖解决方案 为了减少兴奋毒性,准备一种基于胆碱的溶液,在解剖过程中使用(如mM):87 NaCl,2.5 KCl,37.5胆碱氯化物,25 NaHCO3,1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 25葡萄糖。在实验前不到1周准备解剖溶液。 录制解决方案 制备?…

Representative Results

含有视网膜原性和皮质化通路的dLGN切片制备显示在4x目标下(图2)。视网膜结节细胞的斧头在视道中捆绑在一起(图2)。刺激移液器被放置在视道上,分别诱导视网膜突变性突触介导电流(图2A)和细胞核神经酸酯,分别诱导皮质原化突触介导电流(图2B)。值得注意的是,从皮质神经元到LGN神经?…

Discussion

我们描述了基于先前发布的方法3的改进协议,它允许研究释放视网膜突变性突触的高概率和从同一切片释放皮质化腺突触的低概率。这是非常重要的,因为这两个输入相互作用,以调节视觉信号传输:视网膜-光化输入是继电器神经元的主要兴奋驱动,而皮质性输入作为调制器,这影响视网膜抗原传输的增益,影响中继神经元的状态。正如所料,我们观察到视网膜增生和皮质原酸突触显示不…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作由德国研究基金会(DFG)在协作研究中心(SFB)1134″功能组合”(J.v.E.和X.C.)和研究赠款EN948/1-2(J.v.E.)资助。

Materials

Amplifier  HEKA Elektronik EPC 10 USB Double patch clamp amplifier
Biocytin Sigma-Aldrich B4261-250MG
CaCl2 EMSURE 1.02382.1000
choline chloride Sigma-Aldrich C1879-1KG
Confocal Laser Scanning Microscope Leica Microsystems TCS SP5
CsCl EMSURE 1.02038.0100
Cs-gluconate Self-prepared Since there was no commercial Cs-gluconate, we prepared it by ourselves 
D-600  Sigma-Aldrich M5644-50MG methoxyverapamil hydrochloride
D-APV  Biotrend  BN0085-100 NMDA-receptor antagonist
Digital camera for microscope Olympus XM10
EGTA SERVA 11290.02
Forene Abbvie 2594.00.00 isoflurane
Glucose Sigma-Aldrich 49159-1KG
HEPES ROTH 9105.2
High Current Stimulus Isolator World Precision Instruments A385
KCl EMSURE 1.04936.1000
MgCl2 EMSURE 1.05833.0250
Micromanipulators Luigs & Neumann SM7
Miroscope Olympus BX51
mounting medium  ThermoFisher Scientific P36930 Prolong Gold Invitrogen
NaCl ROTH 3957.1
NaH2PO4 EMSURE 1.06346.1000
NaHCO3 EMSURE 1.06329.1000
Pipette Hilgenberg 1807502
Puller Sutter  P-1000
razor blade  Personna  60-0138
Semiautomatic Vibratome Leica  Biosystems VT1200S
SR 95531 hydrobromide  Biotrend  AOB5680-10 GABAA-receptor antagonist 
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Tags

Keywords: ElectrophysiologicalRetinogeniculateCorticogeniculateSynapse FunctionLateral Geniculate NucleusRetinal Ganglion Cell AxonsRetinal Geniculate SynapsesCortical Geniculate SynapsesBrain SliceDissectionRecordingVibratomeForebrainCerebrumOlfactory BulbMidbrainHemispheresCutting StageBuffer Tray
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Citazione di questo articolo
Chen, X., Wang, D., Kegel, M., von Engelhardt, J. Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function. J. Vis. Exp. (150), e59680, doi:10.3791/59680 (2019).
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