修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像
Summary
利用多角度切割鼠标小狗大脑，我们在提高先前描述的急性脑切片捕捉之间最重要的听觉脑和前脑结构的连接。
Abstract
The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.
Introduction
在听觉系统，虽然有感官周边和下丘之间的信息实质性加工，有相当大的附加的处理之前到达听觉皮层。我们所知甚少有关如何处理已完成，因此一些关于这种转变让大脑解释传入的感觉信息。除嗅觉，每个感官具有与最初被中继高保真其中下降的信号上升到皮质外设信号非常相似的组织。皮质然后发送突起到下部结构来进一步调节所述输入信息。这种复杂的系统进行了研究，以各种方式在体内以及在一些体外制剂。在前者中，所有的连接都完好，使研究者探测任何组连接，同时控制感觉输入和测量输出在任何给定的区域中。采用这种方法，很少有无法控制的种类繁多的其他投入，包括其他感觉输入，觉醒和重视，以一种强烈的复杂的输出便出现在体外 ，大脑切片被切断捕捉到一个单套的预测，或者两个相连的脑区，这使研究人员，以刺激和评估各种传入或脑区。这些通常是要么丘脑皮层或tectothalamic片，其中任一输入到丘脑或丘脑和其输出到皮层被保留2-5。这些制剂允许各种各样的药理，电气和光遗传学操作。然而，只有两个脑区，它们主要评估的信息的传送，并且缺乏以评估信息的改造，因为它通过丘脑的能力。另外，网状丘脑突起，其可在关注调制发挥作用6-9是公关ESENT在此片。在这里，我们展示的改进在我们之前的准备1，允许各种投入到丘脑研究者控制给予的丘脑盖茨和过滤信息如何以独特的视角。我们夫妇这种新颖的切片准备与黄素蛋白自发荧光成像来评估片连接和大规模活化分析，在丘脑神经元群体分析钙成像，和单细胞记录以测量的各种输入上的单细胞水平的影响。
为了协助维护这些应用广泛的联系，我们还开发了一些垂直切片锚（又名“竖琴”）的修改举行的脑切片的地方，桥梁，以提升切片增强灌注。竖琴被设计成修改马蹄形以包围切片，并允许可定制的连接点为竖琴字符串。三字符串附，使得i）一种位于沿水平方向内侧边缘的切片，ⅱ）从IC到AC的尾部边缘的尾部边缘上的一个延伸，并且iii）一种从斜内侧缘的片的延伸到的区域喙到AC（ 见图 1A）。小凹口在帧胶合（与氰基丙烯酸酯胶）的琴弦允许有减少量的压力中的片上，以帮助保持片完整性的（参见图1B）。通过使用三维印刷，我们能够定制设计竖琴我们独特的规格，以及其允许的上方和下方的组织的人工脑脊液（ACSF）理想的流量桥。这也保持着大面积的光穿透组织的膜片钳电。
Protocol
Representative Results
Discussion
This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.
Materials
| High sucrose cutting solution | in mM: 206 sucrose, 10.0 MgCl2, 11.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 2.5 KCl, pH 7.4 | ||
| Low calcium aCSF | in mM: 126 NaCl, 3.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1.0 CaCl2, 2.5 KCl, pH 7.4 | ||
| aCSF | in mM: 126 NaCl, 2.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 2.5 KCl, pH 7.4 | ||
| Stimulus Isolator | World Precision Instruments | A360 | |
| DMSO | Life Technologies | D12345 | Lot: 1572C502 |
| Fura-2AM | Life Technologies | F1201 | Lot: 144912 |
| Pluronic F-127 | Life Technologies | P3000MP | Lot: 1499369 |
| Large culture dish | Fisherbrand | 08-757-13 | 100x15mm culture dish |
| Small culture dish | Falcon | 353001 | 35x10mm culture dish |
| Raised culture membrane | Millicell | PICMORG50 | Used to maintain oxygenated fluid perfusion on both sides of slice. |
| Flavoprotein imaging fluorescence cube | Olympus | UMNIB | 470–490 nm excitation, 505 nm dichroic, 515 nm emission long pass. We have found that virtually any green fluorescence protein filter cube will work here. |
| Calcium imaging fluorescence cube | Omega Optical | BX-18 | XF1005 365nm exitation, XF2001 400nm dichroic, XF3080 510nm emission |
| Agar for blocking brain | 3% by weight in water | ||
| Viper si Stereo Lithography Apparatus | 3D Systems |
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