突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

Published 3/23/2011
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Summary

本文概述了程序,准备从老鼠和转基因小鼠海马突触改变与大脑衰老和与年龄有关的神经退行性疾病如阿尔茨海默氏症,相关的研究。

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Mathis, D. M., Furman, J. L., Norris, C. M. Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology. J. Vis. Exp. (49), e2330, doi:10.3791/2330 (2011).

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Abstract

啮齿类动物的海马脑片制备也许是最广泛使用的工具为调查哺乳动物突触功能和可塑性。海马可快速,轻松地从大鼠和小鼠中提取和切片仍然可行含氧人工脑脊液小时。此外,基本electrophysisologic的技术很容易应用到海马突触功能的调查,并提供了一​​些认知障碍的最好的生物标志物。海马脑片是在学习和记忆有关的突触可塑性机制的研究,特别是流行的。诱导长时程增强和突触效能的抑郁症(LTP和LTD)在海马(或缺乏)的变化是经常被用来描述神经表型的认知受损的动物和/或评估行动的机制益智化合物。本文概述了我们准备从老鼠和转基因小鼠海马突触改变与大脑衰老和阿尔茨海默氏病(AD)1-3相关的研究中使用的程序。使用的老年大鼠和AD模型小鼠可呈现出一套独特的挑战,习惯于在他们的研究中使用年轻的大鼠和/或小鼠的研究人员。老年大鼠有较厚的比年轻大鼠和小鼠的头骨和更严厉的结缔组织,可延缓脑提取和/或夹层,并因此否定或夸大真正的突触功能和可塑性的年龄差异。老龄化和淀粉样蛋白的病理还可能加剧在解剖过程持续的海马损伤,再复杂的生理评估得出的任何推论。在这里,我们讨论了在解剖过程中采取的步骤,以尽量减少这些问题。在“健康”和“不健康”的片从大鼠和小鼠获得的突触反应的例子,以及有代表性的突触可塑性实验。在这些动物模型(如录音解决方案组件,刺激参数)的突触功能的其他方法因素可能产生的影响进行了讨论。虽然本文的重点是对老年大鼠和转基因小鼠,新手使用切片生理,要找到足够的细节,在这里开始自己的研究,使用各种啮齿类动物模型。

Protocol

1。准备冰冷含氧人工脑脊液(学联)

  1. 准备2“的Ca 2 +”学联大号。在2L的锥形瓶中,加无菌双蒸H 2 O约1.5升,并开始搅拌小的轰动板大力。添加以下学联组件(毫米):124氯化钠,氯化钾,1.25 KH 2 PO 4,2,26硫酸镁碳酸氢钠3,和10葡萄糖(见表1)。带2升的体积, 蒸馏水H 2 O
  2. 使用一个水族馆起泡和油管连接到95%的O 2 / 5%的CO 2空气罐,氧气学联约20-30分钟大力。检查pH值,如有必要,调整至7.4,用NaOH或HCl。
  3. 一个单独的锥形瓶中倒入750毫升含氧的Ca 2 +免费学联,用封口膜覆盖开幕,并转移ultrafreezer(-80℃)20-30分钟。此媒体将用于解剖的大脑和急性海马*.新增2毫米氯化钙2余下的1.25升容积的学联#炒匀,并恢复95%的O 2 / 5%的CO 2充氧。此媒体将被用于存储解剖后,为灌注过程中的电生理记录片大脑切片。
    *冰冷的Ca 2 +自由的媒体是用于代谢缓慢,并尽量减少夹层的Ca 2 +依赖的兴奋性毒性。
    #变化的Ca 2 +在衰老过程中的失调和AD可以有一个重大影响诱导的Ca 2 +依赖的突触可塑性 4-9 。部分原因可能是由于使用不同学联的Ca 2 +:镁+比值在片录音(见2,4,10)有限公司的年龄差异上的矛盾的报告。 重要性+调节和学联的Ca 2 +在老龄化的研究水平是在讨论中更详细的解决。
  4. 虽然的Ca 2 +免费学联冻结,准备维护脑片电生理录音之前和期间的控股室*我们使用一个自定义宏控股室包含四个个人microchambers(见图2)。 macrochamber是用无菌填充,含氧H 2 O和microchambers基本上都是伸出水面以上的岛屿。在每个microchamber,切片休息拘捕的含氧学联浅池插入。片完全淹没,但坐在一个接口与空气加湿。绝缘的加热元件内的macrochamber允许的温度调节。
    控股商会的几个品种也市售(如华纳仪器使得一个浸入式“前商会#BSC - PC),并应适合在老龄化和转基因小鼠切片研究。在紧要关头,片可维持学联填充在一个小培养皿菜的几个小时。氧气输送管的一个小孔,在培养皿盖子,要小心,氧气气泡不身体搅拌片。切片将得到足够的氧气输送管是提高公正以上学联表面(气散,导致在液体表面的压痕)。

2。老年脑拆卸和海马解剖(> 20月龄大鼠)

  1. 准备清扫面积*旁边的一个大水槽(见图1和表2)。放置在水槽中的小动物断头台和手术器械和材料去除脑和海马清扫,包括一个折叠的纸巾,#11手术刀刀片,beebee剪刀,骨rongeurs,“海马工具”(一个专门的双锅铲从精细的手术工具),小的手术剪刀,薄薄的双端锅铲巴斯德吸管,塑料,直径110毫米的滤纸过滤纸,有盖的100毫米的玻璃培养皿中充满冰和塑料勺。另外,保持一个塑料袋附近的胴体处置。
    脑提取应尽快完成,所以这是一个好主意精神“穿行”的程序和躺在仪器的使用顺序。
  2. 从冰箱中取出的Ca 2 +无学联。媒体应部分,不完全冻结。一个玻璃烧杯中倒入约50毫升的学联,封口膜覆盖,放在旁边的清扫面积。该媒体将被用来简单地存储在大脑,一旦它被删除。其余的Ca 2 +自由的媒体将被用于填补片准备Vibratome水库。这种介质可以复氧,或简单地用封口膜覆盖和在冰箱放置在4 ° C。
  3. 安乐死的大鼠,使用机构的动物护理和使用委员会(IACUC)批准的方法。我们的动物被放置在一个有机玻璃小室,正逐步与100%的CO 2填充。意识丧失,一般发生在五分钟内,并通过反射活动的情况下证实下面的一个脚趾头掐。
  4. 杀头大鼠延髓第一颈椎和折叠纸巾放在头。使用11#手术刀,迅速在中间的起点附近的鼻骨和尾端枕骨头皮切口。一定要削减完全通过皮肤肌肉,充分暴露上的头骨背表面的缝合线。
  5. 穿过beebee剪刀的scull板。年轻的大鼠和小鼠,可迅速取出头骨与单独使用的骨rongeurs。然而,老年大鼠比年轻成年大鼠和小鼠,可以使这个过程很难厚的头骨。我们发现,穿过头骨,极大地方便了与rongeurs骨去除,减轻大脑损伤,并节约了宝贵的秒。随着老鼠的坚决柜台上方的头部,将降低到优越的枕骨大孔的头骨后部地区beebee剪刀纯粹的前沿。保持较低纯粹坚决反对头骨的内表面(远离大脑)*通过枕板切割,然后沿中线缝合壁层板。 rostrally继续,直到你通过正面的颅骨板切割。
    *这是极为重要的,压力是针对远离大脑,以防止不慎测量。
  6. 独立的枕叶,顶叶,从大脑颞颅骨板。继续坚定地抱着头部稳定,并充分利用台面和幻灯片的rongeurs底部颚下的左顶叶板保持对头骨的内表面的压力。下一步,挤压rongeurs颌骨向上和向你滚你的手腕拉从大脑的顶叶和枕叶板距离。这应该公开的左半球的背水面。如果有必要,使用rongeurs以及删除左侧额叶板。重复这个过程中,另一半球。一旦板块流离失所,快速检查任何硬脑膜问题可能附加颞板块和整个大脑的表面伸展。轻轻拉动这些rongeurs或用剪刀剪开距离*现在,幻灯片之间的大脑和右颞板的rongeurs上颚,再次保持向着头骨和大脑远离压力。挤压和扭曲远离大脑颞板。您应该会听到/感受到“紧缩”,你这样做。左侧重复。
    *硬脑膜很难发现的动物,尤其是如果已经transcardially学联灌注。但是,如果硬脑膜不会被删除,他们将切片通过大脑(和最有可能的“海马”),如颞板时,都将被删除的剃须刀。远离截图颞板附近的硬脑膜,将最大限度地减少刺向大脑的可能性。
  7. 提取脑*快速删除的Ca 2 +学联“泥泞”的封口膜。现在幻灯片之间的大脑和底部的颅骨板腹面海马工具的广泛锅铲头,直到它完全是根据大脑。移动刮板横向从一侧到另一侧,然后向前和向后几次断绝完整的颅神经。现在舀大脑海马工具和淹没在Ca 2 +无学联,用封口膜覆盖。让我们一分钟左右的大脑寒意。这是一个时机,清洁断头台,处置的胴体,和重组清扫面积。
    *步骤2.3-2.7,速度是至关重要的。我们尝试在30-35秒内完成这个程序(斩首脑学联淹没)。根据我们的经验,提取到的时间不到一分钟出现海马脑片的健康产生不利影响,特别是对老年大鼠。使用这些程序,我们已经看到在我们研究的中年和青年大鼠之间的提取次数没有差异。
  8. 提取的海马。冰冷的培养皿的盖子上的Whatman滤纸和打击与学联的纸张,使用的塑料移液管。检索学联脑挫伤的Whatman纸用勺子和地方。使用的手术刀刀片,去除小脑延髓额叶和大约四分之一。通过intrahemispheric裂运行的手术刀刀片完全分开的两个半球。放置一个半球放回学联泥泞和“立场”上剖析阶段,如额叶冠状面朝下了。你应该很清楚地能够区分脑干和中脑覆皮质(粉色/浅灰色)(白)。定位在中脑的优势和劣势丘,这些看起来像两个白色的“旋钮”,将在“顶”的大脑在这个方向。使用的手术剪,轻轻地抱在中脑和幻灯片的差距称重锅铲之间的丘和新皮层。轻轻地,继续向下滑动刮板和拉脑干/脑/丘脑离露出里面的侧脑室和海马的内侧表面。使用锋利的刀刃锅铲顺利切断穹窿;白色的纤维束位于前/背海马的部分。用剪刀,轻轻地继续拉脑干/脑/丘脑距离,没有完全切断从大脑的休息。皮质与海马现在应该从脑干奠定自由*.下一步,将解剖的阶段,让你在内侧的海马和覆皮质就好像它是一个矢状切面(减去丘脑)。您现在应该看到的白伞的纤维,在海马的底部形成了一个这架飞机的浅双曲线。使用的伞下面的差距的塑料移液管,轻轻喷一些学联帮助单独从皮质的海马。锅铲轻轻滑入这种差距,这种锅铲长边平行与海马长轴。一旦锅铲是完全根据海马,按住/脑/丘脑脑干坚定地用剪刀和卷锅铲从你的身体从物理上独立的休息大脑的海马。一旦海马是免费的,轻轻地修剪了任何剩余的皮质,血管,和白质。舀到远侧解剖阶段少量的学联泥泞。轻轻的位置旁边的烂泥和扑灭学联使用的塑料移液管几毫升的海马。接下来,删除另一半球和重复清扫。
    *您可能需要的剪刀剪断了额外的结缔组织,白质,或血管,防止脑干皮层的分离。您的剪刀点海马将会非常接近,所以一定要提供非常精确的剪(锋利的剪刀是必须的)。

3。在敬老院的转基因小鼠的脑拆卸和海马解剖

  1. 安乐死和杀头的鼠标和一个正中切口在头皮上,在2.3节中所述。老鼠有薄得多的头骨比大鼠,从而大大简化了大脑提取。通过颅骨,用剪刀切割,因此没有必要。使用与较小的下颚(见表2)骨rongeurs绝尘而去的枕叶,顶叶,和颞颅骨板。与大鼠,记得使用控制运动和坚决的rongeurs下颌和头骨的内表面拉成从大脑删除板块。一旦板块都将被删除,使用海马工具割断剩余的颅神经和大脑舀入冰冷的Ca 2 +免费学联的窄端。
  2. 准备的大脑切片。由于鼠脑的体积小,解剖海马,可以是一个有点更具挑战性的(但肯定是可行的),比使用老鼠时。因此,为了使事情变得更容易,我们删除额叶小脑和延髓提示,但不剖析出的海马。相反,大脑半球物理上分开,用手术刀刀片和左完好Vibratome切片(见下文第4节)。

4。节到用振动切片机(Vibratome)的脑切片组织和转让控股厅*

  1. 填写Vibratome水库与冰冷的Ca 2 +无学联,这样的切割阶段和刀片完全淹没。为了使大鼠切片,用手术刀刀片切断每个海马延髓和尾鳍提示。这些裁员将让你像两列垂直位置的海马紧密地结合在一起。这效果最好的,如果每个海马齿状回正面临着一个和CA3区在同一方向的导向。为了使鼠标片,垂直位置每个半球额叶朝下紧密地结合在一起。胶到安装块和切片阶段的Vibratome转移的脑组织。我们通常会准备〜400 UM节突触生理学实验。较薄,应准备将进行荧光成像(如调查 Ca 2 +水平和瞬态) 。收集与宽口吸管或油漆刷#片,并转移到一个小的培养皿中含有冰冷的Ca 2 +免费学联。
    *使用一个Vibratome最大限度地减少片的上下表面的损害,是绝对需要近片表面的细胞(如电压钳和Ca 2 +影像学)分析研究的建议。然而,更便宜的替代品,适合用于发电外生理学实验片。我们用一个小的重力控制菜刀2,11和McIlwain组织斩波器具有良好的成功12。对于THIS程序海马是放置在一个演出,并与一个垂直菜刀切片。画笔是用来传输片(一次一个),一个小的控股菜弥漫冰冷的Ca 2 +免费学联。这种方法的一个问题是,海马之间移动造成不均匀的部分印章。此外,要小心删除尽可能多的白色物质,尤其是尽可能多的血管,前斩尽可能。这种材料将继续坚持刷,刀片,或者,使得切片转移非常困难,并增加组织的拉伸或损坏的概率。
    #当使用刷子,尽量休息整个刷毛片的长度,明智的。环绕着画笔笔尖片可能会导致不必要的组织伸展。
  2. 将脑切片控股室,他们在那里含氧的Ca 2 +含学联沐浴。逐步实现室的温度从27 °到32 ° C(1 °每五分钟)。允许片电生理实验前为1-1.5 h孵化。

5。引出并记录CA3 - CA1区突触反应

  1. 对于急性切片的基本外录音,您的电车站,将需要包括*:录音室;灌注系统;> 4X放大倍率能力显微镜;录音,刺激,和接地电极;宏观和micromanipulators一个刚性抗振表的顶部和法拉第笼;刺激;放大器和模拟到数字(A / D转换器)转换器,示波器(最好);和相应的数据采集软件的个人电脑。
    *克尔科学仪器提供了一个梦幻般的和廉价的电,进行各种基本的片电生理实验系统(即克尔组织录音系统)。该系统具有占地面积小,便携式刺激器和放大器,可以在一个标准的实验室工作台的使用,而不需要一个庞大的法拉第笼。
    当然,也可用于脑切片执行许多精心制作的电生理和成像技术,这将需要额外的设备和材料。举例来说,我们主要的电车站,包含一个快速电压和电流钳位功能,荧光灯照明系统,数码相机的放大器。这台用于大脑切片,以及膜片钳切片和细胞培养3,12,13录音和荧光成像外的现场录音。的设备和部件的完整清单,请参阅表3。
  2. 使用宽口吸管或小油漆刷,传输一个或多个切片*允许它适应环境刺激/录音前10-15分钟的录音室。对于我们的研究,切片被淹没在学联和休息扣除华纳仪器插入到一个RC - 22室(见图3)。学联是地心吸力通过调整流量的调节,并预热至32 ° C的内联前到达录音室的微加热器。一个中央真空线是用来删除ASCF。
    *许多不同品种的潜水和界面风格的录音商会均为市售。我们观察,切片表现出更强劲的突触反应接口室(即切片坐在一个接口与空气加湿和学联)2,11 。然而,响应一般比较稳定时,片淹没在学联。在淹没腔药物灌注,也更有效率。
  3. 位置的刺激和记录电极。随着刺激或刺激隔离器开启,但输出拨打下降到0,位置比在地层radiatum CA2 CA3区边界附近的区域(见图4)切片的刺激电极。我们使用了双绞线的铂铱丝,提供50-100我们的双相脉冲CA3区侧枝(SC)的纤维。使用铂铱丝和双相脉冲可以帮助减少电极极化。 CA1区地层radiatum记录电极下,刚刚破片表面。我们的记录电极,Ag / AgCl电极丝是在学联填充玻璃微管(TIP〜7MΩ的电阻)。转到刺激的输出到一个适度的水平(我们设置我们的刺激隔离〜150μA),并开始管理刺激脉冲和收购活动,从Axon仪器,公司使用采集软件,如Clampex,慢慢放下小的刺激电极直到刺激artificact间隔记录CA1区。下一步,继续缓慢降低间隔的刺激和记录电极,同时收购,直到纤维凌空(FV)和兴奋性突触后电位(EPSP)的振幅达到最大水平(见图4B)CA1反应。
  4. 建立突触强度曲线和调查突触可塑性。要产生突触强度曲线,提供刺激脉冲到SC,在越来越高的强度和记录日发送相应的活动,在CA1区。刺激的强度级别使用的范围和数量可能会有所不同,但应足以产生一个S形曲线时,对公允价值或EPSP值(图5)绘制。公允价值的幅度提供了一个可靠的估计,而激活的突触前纤维的比例EPSP的斜坡提供的CA3 - CA1区突触电流的未受污染的措施。因此,绘制对公允价值的跨刺激的强度水平EPSP的斜坡反映每激活传入的EPSP的大小和CA3 - CA1区突触强度提供了一个优良的估计。通常情况下,突触强度的水平显着降低的老年大鼠和APP/PS1小鼠,相对年轻的大鼠和年龄相匹配的野生型小鼠的,例如,看到4,14 CA1区的面积。
  5. 从统计分析(见图4C)切片显示在突触强度曲线的健康状况不佳的迹象(即最大的EPSP的幅度<1毫伏),或过度兴奋(即2个或更多的人口峰值的出现)被排除在外。我们发现,这些片很少表现出跨60分钟内和/或显示高度可变的反应诱导突触可塑性的稳定的反应。值得指出,“不健康片”弥补所有切片转移到录音室只有约10-20%。此外,在我们的经验,确定一个不健康的片的频率是不同年龄,物种和基因型非常相似。
  6. 诱导长时程增强(LTP),切片或长期抑郁(有限公司)。 LTP和LTD持久的增加(LTP),减少(有限公司)和突触功能的突触激活不同的图案。人们普遍认为这两个进程,反映学习和memory15的重要机制,并提供调查的细胞机制的神经功能障碍和/或评估为缓解记忆障碍和神经退行性疾病16的药物战略的有益成果的措施。
    突触可塑性的实验,复位所有片为1 mV *刺激的强度,并开始在0.033赫兹的频率基准的刺激。 EPSP的斜率值应该是稳定的,至少在20分钟前诱导LTP或LTD。在此期间,密切监察EPSPS和复位的刺激强度,如果斜坡波动超过10%,并开始一个新的基线。使用一个次列车为100 Hz刺激或多个短时间(约10个脉冲),给予每200毫秒,100赫兹刺激诱导LTP的。对于公司的诱导,在1 Hz的频率提供900刺激脉冲。 LTP或LTD的诱导后,收集额外的60分钟或更多的突触反应。 EPSP的斜坡前和高/低频率刺激后60分钟获得的值进行比较,以确定存在LTP或LTD。
    老年大鼠和小鼠APP/PS1往往表现出缺乏LTP和增强有限公司(见图5),这些变化已建议作出贡献,这些动物模型6,16受损的认知。然而,与APP/PS1小鼠,老年大鼠的LTP / LTD的变化是整个实验室的变量。老年大鼠一般表现出相似的LTP的比较“激烈”(例如,100 Hz)刺激的反应,以成人的水平,但显示赤字时,温和的刺激参数(即刺激频率较低或更少的刺激脉冲)(审查看到4,616)。此外,包括我们的一些实验室,已观察到的易感性增加 2,17-19岁大鼠有限公司感应,而其他组没有发现任何差异或年老动物的敏感性降低。下面简要介绍(见讨论),微妙而关键的实验协议的差异可能会考虑这些差异。
    *刺激的强度和EPSP的幅度可以影响LTP的诱导和文献中有差异的结果可能是一个重要原因。在讨论中,这将进一步审议。

6。代表性的成果

我们的工作,并从其他小组的工作,建议在星形胶质细胞为基础的炎症信号的变化可能会触发和/或加速衰老过程和AD 13,20,21的神经功能障碍。最近,我们作为端点措施调查的有效性和行动了一些新的抗炎中旬岁APP/PS1小鼠的试剂的机制见22说明此模型)突触强度,LTP和LTD岁的菲舍尔344大鼠。下面提供的结果,获得使用本文中所描述的协议。

其中的新型消炎腺相关病毒(AAV)我们的实验室开发的试剂已在试点研究显示显着增加(P <0.05),突触强度和防止中老年LTP的赤字(P <0.05)(16月龄)APP/PS1小鼠(N = 4-6片每次治疗条件)。代表突触强度曲线和LTP的实验,从两个不同的切片,COLL从相同的16个月大的APP/PS1鼠标ected,显示在图5A - C。从我们的新颖的腺相关病毒治疗的半球中提取一个切片(试剂A),而另一片治疗与控制的腺相关病毒试剂(管制)。 LTP的诱导片在同时使用两个1秒100 Hz的刺激(intertrain间隔10秒)的列车。需要注意的是一个治疗的试剂片的突触强度曲线转移到左侧的控制片,更大的突触强度的指标。另外请注意,典型的中老年APP/PS1小鼠,LTP的腐烂迅速控制片(如23)的基线。相反,LTP的腐烂在与我们的新试剂处理切片。

在第二个最近的一项研究中,我们发现重大有限公司车辆处理的老年大鼠(85%预有限公司基线,P <0.05)。与此相反,没有公司在新型抗炎药物(前有限公司基线的97%,不显着)治疗的老年大鼠观察。突触强度上没有药物的效果进行观察。代表公司,从这个数据集(N = 8-10组大鼠)实验说明在图5D - F。

图1
图1。工具和材料,用于脑清扫,纸巾。 B,手术刀刀片。 C,Beebee剪刀。 D,骨rongeurs(只)。 E,骨rongeurs(小鼠/只)。楼,塑料勺。 G,塑料移液管。 H,海马工具。我,锅铲。 J,手术剪。 K时,玻璃培养皿。

图2
图2。控股自定义的脑片室,macrochamber 。 B,盖子。 C,H 2 O水库硅胶管穿孔。研发,microchamber。 E,学联输送管(聚乙烯)。 F,O 2输送管。 G,温度控制端口。 H,拘捕microchamber插入。

图3
图3。 RC22淹没室 ,录音室。 B,接地电极。 C,穿刺针。

图4
图4。海马脑片的插图和外波形的横向海马电生理实验中使用的部分,卡通。 CA =大角Ammonis。 DG =齿状回。 SC =谢弗抵押品。小号radiatum =地层radiatum。 B,SC(CA3区神经轴突束)的电刺激引起的刺激神器,紧接着人口突触前扣球,或纤维凌空(FV)。公允价值的幅度是SC激活纤维的数量成正比。负相场兴奋性突触后电位(EPSP)斜率直接对应的去极化响应CA1区锥体神经元突触电流的激活,谷氨酸释放从SC终端。 C,重叠的代表外波形记录在CA1区地层radiatum九种不同的刺激强度水平在一个“健康”(左图)(30-500μA),“不健康”,“hyperexcitable”片。五波形平均每级。健康切片动态响应这种刺激范围内,并表现出单一的人口正尖峰(反映CA1区神经元放电)刺激在较高的水平。 RC22淹没室,最大的EPSPS通常范围从1.5至3 MV振幅。不健康片(中间面板)往往表现出一个大型的公允价值,但一个小的最大EPSP(<1毫伏),通常表现出较差的可塑性。 Hyperexcitable片(右图)显示的EPSP的升支的再生两个或两个以上的人口峰值。 hyperexcitable片的反应往往是不稳定和可变有限公司/ LTP的刺激影响。

图5
图5。代表电实验上中旬平均年龄(16 MOS)APP/PS1小鼠和年龄(22 MOS)菲舍尔344大鼠急性片。收集面板AB公司的数据显示与控制腺相关(AAV)病毒治疗的APP/PS1小鼠构造(控制),或​​一个新的腺相关病毒试剂(试剂A)已被我们的实验室集团开发。相对控制片,切片处理试剂A展品的EPSP明显左移:FV曲线(a)更大的突触强度的指标。试剂,一个处理切片也显示了强劲和稳定的LTP(b)在两个1秒,100 Hz的刺激列车交付,同时展品缺乏控制片LTP的,这种动物模型的典型。面板东风从两个单独的老年大鼠慢性(4周)海马汽车或一种新型的消炎药物(药物)灌注收集的数据显示。基础突触强度相对药物治疗的影响(四)。然而,药物的一个非常有效地防止诱导有限公司(五)。面板C和F显示代表EPSP的波形记录个别切片前(前)和60分钟后(POST)交付LTP / LTD刺激。请注意,刺激的工件不显示。

Discussion

在本议定书中概述的步骤将有助于确保至少为迅速有效地开展脑解剖年龄在年轻成年大鼠,。我们还提供足够的细节,对于初学者LTP和LTD成立自己的切片研究。如果老化和突触功能和可塑性AD变化的进一步探索,是您的目标之一,有至少有两个其他的方法问题,上面提到的,值得进一步考虑。首先,一些实验室已经表明的Ca 2 +:Mg2 +的比例在录音学联可以在2,10,24,25海马诱导突触可塑性的一个显着的效果。在哺乳动物脑脊液中 Ca 2 +:Mg2 +的比例大约是(如 26) 。然而,学联的Ca 2 + Mg2 +的比例接近2片突触功能和可塑性研究。在早期的研究中,这种做法很可能是调整,优化诱导的LTP,随后成为日常所有的可塑性研究。然而,这种做法可以在老化和AD的研究问题,因为在神经的Ca 2 +调节的特点差异。具体来说,CA 2 +内流和/或Ca 2 +诱导的Ca 2 +释放是升高的老年大鼠和/或在AD模型小鼠神经元激活3,27-31。有限公司感应是在学联的Ca 2 +水平的微妙的变化特别敏感。我们的协议,采用2mm的钙2 +和2 mM的镁,在公司通常结果年龄,但不年轻的成年动物2,同时研究利用一个钙2 +:镁+比例接近两个,已观察到的成年人强大的公司在该没有年龄差异2,10结合减少有限公司或在32岁的大鼠。这些意见强调需要仔细考虑学联钙2 +和Mg2 +水平进行比较时,在中青年成年动物的Ca 2 +依赖可塑性。

第二个方法问题, 涉及 33突触后膜的去极化和突触强度的差异可能老化/基因型的LTP的强烈依赖。在一个典型的LTP的实验,基线和LTP的刺激强度是一般调整,以生产半最大(或三季度最大)EPSP的幅度。潜在的问题是,通常表明,老年大鼠和APP/PS1小鼠突触强度降低相对年轻和/或野生型对口,这意味着,基线EPSP值也将被更小,老年大鼠和APP/PS1小鼠。较小的EPSPS可能转化为在LTP的刺激少的去极化,导致减少的概率为诱导LTP的33。由于这种潜在的混淆,这是很难以确定是否这些动物表现出吞吐量赤字,可塑性赤字或两者。也就是说,年龄和/或APP/PS1小鼠LTP的诱导机制可能是功能完好(无可塑性赤字),但在这些条件下不够刺激(吞吐量赤字)。这个区别是至关重要的,吞吐量和可塑性机制机制的反应可能非常不同的一个特定的药物治疗。我们尽量减少吞吐量降低的LTP诱导的影响,通过规范的所有片LTP的刺激前EPSP的幅度相同的水平(如为1 mV)。其他策略可能会有效,以及(如电压或电流钳的使用,以平衡各群体之间在膜电位的LTP的刺激),并调查在这些动物模型的LTP时,应考虑。

Disclosures

生产这种视频文章是徕卡Microsystems公司赞助。

Acknowledgements

由美国国立卫生研究院授予AG027297,从肯塔基脊髓和颅脑损伤的研究信托奖,并从Kleberg基金会的礼物支持的工作。

Materials

Name Company Catalog Number Comments
NaCl Fisher Scientific BP358-1
KCl Fisher Scientific BP366-500
KH2PO4 (monobasic) Sigma-Aldrich P5379-100G
MgSO4 Sigma-Aldrich M2643-500G
CaCl2 (dihydrate) Sigma-Aldrich C3306-250G
NaHCO3 Fisher Scientific S233-500
C6H12O6 (dextrose) Fisher Scientific BP350-1
Table 1. Reagents required
Erlenmeyer Flasks Fisher Scientific FB-500-2000 FB-500-1000
Aquarium Bubbler Used for oxygenating media. Available at most pet stores
50 mL Glass beaker Fisher Scientific 02-540G For brain storarge in ACSF
Parafilm Fisher Scientific 13-374-10
Small Animal Guillotine World Precision Instruments, Inc. DCAP-M
Flat paper towel
#11 Feather surgical blade Fisher Scientific 08-916-5B
Beebee bone scissors Fine Science Tools 16044-10
Lempert Rongeurs Roboz Surgical Instruments Co. RS-8321 Use for rats
Friedman-Pearson Rongeurs Fine Science Tools 16020-14 Use for mice or rats
Hippocampus tool Fine Science Tools 10099-15
Spoon A plastic teaspoon will do
Spatula Fisher Scientific 21-401-25A Spatula
Surgical iris scissors Fine Science Tools 14058-09
plastic transfer pipets Fisher Scientific 13-711-43
110mm Whatman filter paper Fisher Scientific 09-805E Whatman cat. 1001-110
Glass petri dish Fisher Scientific
Leica VT1000P Manual Vibrating Microtome Vibratome VT1000P
0.1mm FA-10 Feather S blade Ted Pella, Inc. 121-9 0.1mm FA-10 Feather S blade
Borosilicate Glass Pasteur Pipet (with rubber bulb) Fisher Scientific 13-678-20A For transferring slices: Tip is broken off and heat-polished for larger opening
35 mm Polysterine Culture dish Corning 430588 Used for collecting slices after dissection
Table 2. Tools and materials for dissection
Holding chamber Custom Made
P-97 Horizontal Pipette Puller Sutter Instrument Co.
Vibration isolation table Technical Manufacturing Corp.
Faraday cage Custom Made
Pyrex Aspirator Bottle with Bottom Sidearm Corning 1220-1L
Gravity-contolled IV set with regulator Baxter Internationl Inc. 2C8891
Central Vacuum Line Available in most modern labs
95% O2 / 5% CO2 Gas Mix Scott-Gross Co.
TygonTM Lab tubing For O2/CO2 delivery Fisher Scientific Non-toxic, non-oxidizing, comes in a variety of sizes.
Eclipse E600FN Microscope Nikon Instruments with 10x and 40x objectives, near infared filter, and GFP,DS-Red2 filters
Cool Snap ES Digital Camera Photometrics Cool Snap ES Digital Camera
X-Cite Fluorescent Illuminator EXFO X-Cite Fluorescent Illuminator
Microscope Platform Siskiyou, Inc. Custom assembled
RC-22 Submersible recording chamber Warner Instruments 64-0228 Requires P-1 platform and stage adaptor (Product # 64-0277 From Warner)
TC2BIP 2/3Ch Temperature controller Cell Microcontrols
4 Axis Manual Miniature manipulator Siskiyou, Inc.
Platinum Iridium Wire (0.002 in) World Precision Instruments, Inc. PTT0203
A365 Stimulus Isolator World Precision Instruments, Inc. A365 Stimulus Isolator
Multiclamp 700b Amplifier Axon Instruments
Digidata 1322A A/D converter Axon Instruments
PClamp software Axon Instruments
Personal Computer (Pentium 4) Dell
Table 3. Electrophysiology equipment and materials

DOWNLOAD MATERIALS LIST

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Comments

30 Comments

  1. A great protocol and discussion. What kind of stimulating electrode do you use?

    Reply
    Posted by: Anonymous
    May 3, 2011 - 12:16 PM
  2. Thank you Louise. We use platinum iridium wire to make a bipolar electrode. The last time we purchased this wire we used World Precision Instruments (cat # ptt050²). Let me know if you need any other info

    Reply
    Posted by: chris n.
    May 3, 2011 - 12:54 PM
  3. Great protocol. However, I have got a problem. I can observe huge fiber volleys but with small or no EPSPs. Do you know what could be the problem? Thank you very much.

    Reply
    Posted by: Anonymous
    October 11, 2011 - 10:57 AM
  4. Hi Olivier. Thanks for the kind words. In re to huge FVs and small EPSPs...Sometimes the fix can be as simple as tweaking the placement of your stimulating and recording electrodes (e.g. adjusting the depth and distance between trodes). However, if you've tried this and are still having problems there are a few possibilities. It could be that the tissue is being damaged and/or mishandled during the dissection/slicing procedure. As we mentioned in the protocol above, tissue from old animals and amyloidogenic animals tends to be a little more vulnerable to damage caused by the slicing procedure. If you haven't already, you may try dissecting a young adult mouse/rat (31 C). EPSPs are generally smaller at colder temperatures. 3)Make sure the pH of the incubation/recording medium is between 7.35 and 7.45. In the past, I've found that slices tend to exhibit big FVs and small EPSPs when they're sitting in media with a low pH (<7.3). 4) Make sure the slices are getting oxygenated efficiently DURING incubation, else they will die :). Usually, if oxygenation is good during incubation, but poor during the recording, the slices start off looking good but then become hyperexcitable as the experiment progresses. I hope this info helps. If you've tried all these fixes with no luck, feel free to email me.

    Best,

    Chris

    Reply
    Posted by: chris n.
    October 11, 2011 - 12:52 PM
  5. Hi Chris,

    Thank you very much for your response but I think still need to bother you again. Indeed, it dŒs not seem to be related to the age of the animals. I think my slice are ok even if I use a McIlwain slicer. Indded, I had some responses last week (from 0.² to ² mV with immerged slices). Artefacts and fiber volleys were small compared to the responses. Now I can't see anymore proper responses (or very rarely) and as I said before, the amplitude of the fiber volley is huge (0.5-² mV) even for small stimulations. Moreover, it has two components a positive and a negative one. The artefact of stimulation became incredibly huge e.g 35 mV for a 100 us stimulation at 500 uA. Would you have any suggestion? I will test my headstage with the model cell. Thank you very much.

    Best regards,

    Olivier

    Reply
    Posted by: Anonymous
    October 13, 2011 - 2:42 PM
  6. Hi Olivier, sorry for the late response. If you wanted to send me a picture of your EPSP waveforms to my email address (should be listed somewhere above or on the .pdf), I'd be happy to take a look at them. If you send this to me, please include information on the type of stimulator unit and electrodes your using as well as your ACSF composition.
    Best,
    Chris

    Reply
    Posted by: chris n.
    October 18, 2011 - 12:12 PM
  7. Dear Chris,

    I have finally found the solution. Everything is working perfectly now. It was an issue with the glue I used for my holding chamber. Thank you again for your precious help.

    Kind regards,

    Olivier

    Reply
    Posted by: Anonymous
    October 19, 2011 - 5:18 AM
  8. Dear Olivier,
    I am having the exact same problem as you. Would you please be more specific about how you were able to correct it? It would be so helpful!
    Best regards,
    Cristiane

    Reply
    Posted by: Cristiane T.
    July 12, 2012 - 11:58 AM
  9. Dear Cristiane,

    As explained in me previous message my problem of poor viability and small responses was related to the holding chamber to keep the slices at 35°C before experimenting. However, low viability can be due to many other reasons. You should work fast, in presence of AMPA and or NMDA blocker (or low sodium solution). You should avoid to fold the hippocampal tissues etc....

    Hope it will help.

    Reply
    Posted by: Olivier P.
    July 16, 2012 - 4:12 AM
  10. Hi every body, i was really so glad when i see your web site, and i want offer my thanks for your great site.
    i am from shefa neuroscience research center in iran where managed by proff. ALI GORJI who one of the first person worked on spreading depression in the world.
    i am working on Spreading Depression too and use LTP technique.
    please contact with me by e-mail for Future cooperation.
    Best Regard
    ahmad ali

    Reply
    Posted by: Anonymous
    November 18, 2011 - 10:56 AM
  11. Hi, I got a problem with my hippocampal slice recording. When I just transferred my slices from that incubation beaker into recording chamber, a large fEPSP response can be recorded, but after sitting in that chamber for a while, the response tended to become smaller even disapper compared with that when slices were just put in chamber. I have tried to adjusted the rate of oxygenation and temperature to ensure sufficient oxygen supply and correct temperature. I can garantee that oxygen supply has no problem and temperature is in normal range (²6 -30 C). But the problem is still there. Somebody told me that is cause by a "heat shock" when slices are transferred to a warmer enviornment. Can you help to resolve this problem?

    Reply
    Posted by: Anonymous
    January 13, 2012 - 7:03 PM
  12. hi, you should put your slice in chamber(²8- 30c) for 1 hour after you get slice. And after that you should transfer your slice to second chamber( LTP chamber) in 3²c.

    Reply
    Posted by: Anonymous
    February 3, 2012 - 11:48 AM
  13. Hi,

    I suppose you can't record at 35°C, don't you? May be the problem is that your slice moves slightly during the recording. Did you try to change the position of your recording electrode to see if you can recover a response with the initial amplitude?

    Olivier

    Reply
    Posted by: Anonymous
    January 17, 2012 - 1:21 PM
  14. Yes, I did. I move the recording electrode to several different positions trying to elicite a good amplitude. But the problem is still there. I have noticed that after sitting in the recording chamber for a while, the slices tended to lost activity. No matter where you put the recording electrode, you cannot recover a good amplitude as the initial one. I suspected that was caused by a slow flow rate, which is 1.5-² ml/min in my chamer. My chamer is different from others, because I currently use a blind method to record fEPSPs and eEPSCs. So my chamber is bigger than others with approximately 1-² ml volume for fluid exchange. I am trying to increase that flow rate to ².5-3 ml/min to see what will happen.

    Reply
    Posted by: Anonymous
    January 17, 2012 - 3:26 PM
  15. Hi, sorry to hear about the problems you're having. Are your slices submerged in the recording chamber or at an interface with the air? Is your stimulus artifact changing, or just the synaptic response? What about the fiber volley, dŒs it die too? How do you know that the oxygen levels in your media are adequate? If your recording chamber is wide and your bath shallow, that would provide quite a bit of surface area for the oxygen to escape. Testing the bath pH with a micro pH meter would help determine if O² levels are stable. If you're worried about heat shocking the slice when you transfer it to the recording chamber, you could try slowly bringing the temperature of the media in your holding chamber up to recording temperature during the incubation period (maybe raising it 1 or ² degrees every 10-15 min).

    Reply
    Posted by: chris n.
    January 17, 2012 - 3:56 PM
  16. Hello again, I want to know the distance between stimulating electrode and recording one in your experiments. Some people put them in a very short distance (²00-300 micrometers). In my experiments, I usually put the stimulating electrodes at the initial part of the schaffer collateral fibers in CA1 area, and put that recording ones at a site as far as possible to avoid the large artifacts, but sometimes I was unable to get a stable LTP after HFS. I believe the reasons for that might be that many neural circuits are activated during HFS including inhibitory ones, because more circuits can be activated simultaneously with the increased distance. Is that correct?

    Reply
    Posted by: Anonymous
    January 25, 2012 - 5:03 PM
  17. I need more detail, since I am still facing some problem.

    Reply
    Posted by: Zaman K.
    February 9, 2012 - 2:38 AM
  18. Finally, I found out the problem that leads to loss of viability of the hippocampal neurons in my experiment. It was caused by my chamber, which is so different from others. It has a very large volume, with a large surface. So I was assuming that my problem was caused by the anoxia resulted by rapid diffusion of ACSF into the large shallow surface. I already increase the flow rate to ².5 ml/min in my previous chamber, but still had that problem. So I made a polycarbonate slice chamber according to Warner company's criteria. Now I can get a pretty stable response throughout my experiment.

    Reply
    Posted by: Anonymous
    February 16, 2012 - 2:02 PM
  19. What is the significance of air interface vs. submerged slices, is one better than the other?

    Reply
    Posted by: Anonymous
    February 16, 2012 - 2:38 PM
  20. BTW, the criteria to judge the anoxia is the production of bubble in the chamber. If you see the bubble present in the chamber, that means the ACSF is oxygen-saturated. Heating makes the extra oxygen escape from the solution. If not, the ACSF is not saturated by oxygen.

    Reply
    Posted by: Anonymous
    February 16, 2012 - 3:10 PM
  21. Good for morale to read that I'm not the only one having problems with acute slices. Due to the comments above I'm going to try a few new things with my set-up. It MIGHT just be the oxygen-saturation, since I pre-heat the ACSF to 4² Celsius, trying to record at ~34 Celsius AND have a relative large chamber. Temperatures are experimental variables of the design.

    Reply
    Posted by: Anonymous
    February 28, 2012 - 11:15 AM
  22. Increasing the perfusion rate to 4 ml/min and changing to a bath that is diamond shaped and has a far lower volume were the solution. Getting a decent signal is still something of a black magic. =/

    Reply
    Posted by: Anonymous
    April 13, 2012 - 7:14 AM
  23. Thank you for sharing your protocol - excellent video.

    Is there an advantage to dissecting out the hippocampus before slicing, as opposed to slicing the whole brain (or one hemisphere of the brain)?

    If I am not mistaken, you cite "netting" in the bottom of the recording chamber. DŒs that mean that the slice is suspended off of the bottom of the chamber?

    Is the resistance of the recording pipette a critical factor?

    Thank you,

    Reply
    Posted by: Anonymous
    March 24, 2012 - 5:33 AM
  24. Hi Chris, sorry for the late reply. For rats, whole hemisphere slices are fairly large and can be a bit of a problem if you have small recording and holding chambers. Also, the hippocampus in an adult rat is fairly easy to dissect away. For mice, on the other hand, the hippocampus is very small and a little trickier to dissect out (though it can certainly be done effectively). You can therefore save time by simply slicing the whole mouse hemisphere into sections. And unlike the rat, whole hemisphere mouse slices aren't so big that they are difficult to work with and store. In re to netting: yes we do use netting, which raises the slice off of the bottom of the dish. In re to the recording pipette resistance: we typically use smaller tip diameters to minimize damage to the recording region of the slice.

    Reply
    Posted by: chris n.
    June 19, 2012 - 4:36 PM
  25. Hi, Prof. Norris:
    Great protocol to follow and precious comments to refer to. As a beginner to this tech, I have a few questions in my mind.
    1. Some papers claimed that they used sucrose-ACSF(0 Na 0 Ca) as the dissection medium, some even put AMPAR/NMDAR blocker(i.e. Kynurenic acid) and/or ascorbic acid in. According to your experiences, is there any difference btw sACSF and normal ACSF to the slice health? Is postsynaptic blocker and/or ascorbic acid necessary?
    ². In some literatures, they used different ways of anesthetization, such as barbiturates, TCA, ether, isoflurane, halothane etc. DŒs anesthetizing method affect the slice quality and the electrophysiological results? In this paper, you used CO², were there any possibility that the brain might get some anoxia-like influence or damage?
    3. For holding and recording temperatures, I found variable ways in different papers, is there any general principle of how to set the proper temperatures? In your paper, after slicing all the brain sections(put in ice cold 0Ca ACSF temporally), and tranfering them to ²7degree and gradually elevating temperature, is my understanding correct? Could you please tell me how much time will each step take(i mean, "cutting"->"temporally storage"->"recovery solution")?
    4. What are the proper cutting speed (mm/min) and vibrating frequency for hippocampal slices?
    5. what cutting direction is proper? longitudinally from CA3 corner to DG corner or the opposite, or laterally across hippo? or it dŒsn't matter?
    Thanks so much!
    sincerely,
    Hang

    Reply
    Posted by: Hang Z.
    January 30, 2013 - 8:13 PM
  26. I have another question:
    6. In re to literatures, some used bipolar twisted electrodes and some used concentric electorde, do you have some comments to these two types?

    Reply
    Posted by: Hang Z.
    January 30, 2013 - 8:21 PM
  27. i am very interesting the custom brain slice holdiing chamber, would you mind sharing the supplier?
    Thank you.

    Reply
    Posted by: CHIA S.
    March 26, 2013 - 3:23 AM
  28. Hi,

    I was very excited when I came across this video/article today, since my Masters project is going to be entirely based on recording after LTP induction on adult mouse hippocampi in vitro.

    I have just started trying to obtain decent slices, and have only gotten so far as extracting the brain from the mouse with acceptable quality. All that comes after has been a little bit frustrating so far. So, I have a few questions regarding the slicing step itself (we have the very same vibratome at our lab, which makes things easier, I guess!):

    - Every time I try to slice the brain, the blade actually pushes the brain away instead of cutting through it! This leads to either a completely useless slice or not even that, as the blade will only cut the last few milimiters of the brain and yield something not even worth calling a slice! The blades are brand new, but I bought them at a drugstore and they were intended to be shaving blades. Might that be the issue, a blade not sharp enaugh?

    - I haven't yet tried separating the hemispheres. Maybe I should do that!

    - I see you use this little block onto which you glue the brain (hippocampus, in your case). I've been using a sort of plate attached to the vibratome, which gŒs in the same place as the block. Do you reckon the block is a better call?

    - Do you have a better method for drying the cutting chamber after using it than letting the ice thaw and then sucking it up with vacum?

    If there are any remarks you should think of that haven't been shown in the video regarding the actual slicing step, I'd apreciate if you could share!

    Best regards,

    Thomaz Dias
    UFMG - Brazil

    Reply
    Posted by: Thomaz L.
    March 28, 2013 - 5:16 PM
  29. Great information here. I have a quick question about the aCSF containing bottle; would you keep it at room temperature or in a water bath at 34 degrees C?

    I will be recording at 34 degrees with a recording chamber heater and was wondering what would be the best way to hold the aCSF that is coming into the chamber?.

    I know there is an inverse relationship with dissolved oxygen and temperature in water, but would it be better to have it constant in the bottle and chamber or best to have it room temperature in the bottle and warm upto 24 degrees in the chamber?

    Any advice would be much appreciated.

    Minos

    Reply
    Posted by: Minos K.
    July 12, 2013 - 11:50 AM
  30. Hi
    Your projects are amazing. Oh here I can’t help sharing some experience of fabrication after designs done.
    We are working with a OverMolding vendor in China the name is Zetar Industry (http://www.zetarmold.com), Located in Shanghai China (a very big modern city). They are really good.Moving fast and very professional. They helped me with my new design. I asked them to do the injection mold, then Injection Moldingmass production. I was surprised by their rich experience in Insert Molding
    and punched lead time. Guys in Zetar deserve every good word just as you are.

    Reply
    Posted by: info z.
    September 2, 2013 - 11:16 AM

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