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Jun 2016
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Acute Cerebellar Slice Preparation Using a Tissue Chopper
使用组织切碎机快速制备小脑切片   

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Abstract

Acute cerebellar slices are widely used among neuroscientists to study the properties of excitatory and inhibitory synaptic transmission as well as intracellular signaling pathways involved in their regulation in cerebellum. The cerebellar cortex presents a well-organized circuitry, and several neuronal pathways can be stimulated and recorded reliably in acute cerebellar slices. A widely used acute cerebellar slice preparation technique was adapted from Edwards’ thin slice preparation method published in 1989 (Edwards et al., 1989). Most of the acute cerebellar slice preparation techniques use a vibrating microtome for slicing freshly dissected cerebellum from various animal species. Here we introduce a simpler method, which uses a tissue chopper to quickly prepare acute sagittal cerebellar slices from rodents. Cerebellum is dissected from the whole brain and sliced with a tissue chopper into 200-400 µm thick slices. Slices are allowed to recover in oxygenated aCSF at 37 °C for 1-2 h. Slices can then be used for electrophysiology or other types of experimentation. This method can be used to prepare cerebellar slices from mouse or rat aged from postnatal day 7 to 2 years. The preparation is faster and easier than other methods and provides a more versatile diversity of applications.

Keywords: Acute cerebellar slice (小脑快速切片), Cerebellum (小脑), Mouse (老鼠), Rat (), Electrophysiology (电生理)

Background

Acute cerebellar slice preparations, like many other current brain slice preparation techniques, originated from Edwards’ thin slice preparation method published in 1989 (Edwards et al., 1989). In general, cerebellum was quickly dissected and immersed in Ca2+-free aCSF and glued to the stage of a vibratome. After slicing, slices were recovered in regular aCSF for 1-2 h before use (Llano et al., 1991a and 1991b; Kano and Konnerth, 1992). Here we introduce a method adapted from the acute hippocampal slice preparation method used in our laboratory. Freshly dissected cerebellum is rapidly sliced (around 10 s) on a McIlwain tissue chopper without the need for glue or oxygenation during slicing. The slices prepared using this method are as healthy as the ones prepared with a vibratome and can be used for electrophysiology or a variety of other manipulations.

Materials and Reagents

  1. Disposable pipette (VWR, catalog number: 16001-180)
  2. Parafilm (VWR, catalog number: 52858-000)
  3. 60 mm and 100 mm Petri dishes (VWR, catalog numbers: 25373-085 and 25373-100)
  4. Filter paper (VWR, catalog number: 28460-030)
  5. Painting brushes
  6. Gas dispersion tube (Ace Glass, catalog number: 9435-10)
  7. Mice
    1. Adult C57BL/6 (WT) mice
    2. Adult calpain-1 KO mice
    3. Adult calpain-1 PHLPP1 double-KO (DKO) mice
  8. Isofluran
  9. NaCl
  10. KCl
  11. CaCl2
  12. MgSO4
  13. D-glucose
  14. NaHCO3
  15. KH2PO4
  16. Artificial cerebrospinal fluid (aCSF) (see Recipes)
  17. Cutting solution (see Recipes)

Equipment

  1. Chill dissection tools
    1. Angled-tip dissector scissor (Fine Science Tools, catalog number: 14082-09)
    2. Scissor (Fine Science Tools, catalog number: 91401-14)
    3. Curved-tip forceps (Fine Science Tools, catalog number: 11051-10)
    4. Surgical blade
    5. Spoon
    6. Dissecting spatula
  2. Brain slice keeper (AutoMate Scientific, catalog number: BSK4)
  3. Beakers
  4. Ice box 
  5. McIlwain Tissue Chopper (The Mickle Laboratory Engineering, Brinkmann)
  6. Water bath
  7. 95% O2/5% CO2 air tank
  8. -80 °C freezer

Procedure

  1. Prepare 1 L of artificial cerebrospinal fluid (aCSF) (see Recipe 1). 
  2. Pour 500 ml of freshly prepared aCSF into a brain slice container. Put the brain slice container in a 37 °C water bath. Connect the brain slice container to a 95% O2/5% CO2 air tank and oxygenate aCSF. Gas flow pressure is set at around 2 p.s.i.
  3. Pour 100 ml of cutting solution into a 150 ml beaker. Cover the beaker with parafilm, and place it in a -80 °C freezer for 5-10 min, until the solution has a slushy consistency.
  4. Place the ice-cold cutting solution on a large ice tray. Bubble the solution with a gas dispersion tube connected to a 95% O2/5% CO2 air tank for 5 min.
  5. Place the lid of a 60 mm Petri dish on ice. Put a round filter paper onto the lid and wet the filter paper with the cutting solution. This is the brain dissection stage.
  6. Chill dissection tools including angled-tip dissector scissors, large scissors, a curved-tip forceps, a surgical blade, a spoon, a disposable pipette with the tip cut off and two dissecting spatulas on ice. The whole dissection setup is shown in Figure 1.


    Figure 1. Dissecting setup for acute cerebellar slice preparation. A beaker with cutting solution, a dissection stage, and dissection tools were placed on a large ice tray.

  7. Anesthetize animal with isoflurane and decapitate with the appropriate tool (e.g., scissors). Immediately extract the whole brain with dissection tools and place it into the ice-cold cutting solution. This step should be done within 1 min.
  8. Chill the brain in ice-cold cutting solution for 1 min.
  9. Place the brain onto the cold dissection stage. First, cut off forebrain, midbrain, and medulla. Then, cut off pons to isolate the cerebellum (Figure 2).


    Figure 2. Steps to isolate cerebellum from whole mouse brain. Forebrain, midbrain, and medulla were cut off, followed by the removal of pons beneath the cerebellum. Top, the axial view of mouse brain. Bottom, the coronal view of Cerebellum and Pons. Numbers and dotted lines indicate steps and sites of the incision.

  10. Immediately transfer the cerebellum onto the stage of a McIlwain Tissue Chopper. Drain the extra solution around the tissue. Slice the cerebellum into 200-400 µm thick sagittal slices (Figure 3).


    Figure 3. McIlwain Tissue Chopper (The Mickle Laboratory Engineering, Brinkmann)

  11. Move the cerebellum to a 100 mm Petri dish containing ice-cold cutting solution and carefully separate each slice without damaging them using two small painting brushes.
  12. Transfer slices into warm aCSF in the brain slice container using a disposable pipette with the tip cut off.
  13. Incubate slices in aCSF at 37 °C for 1-2 h with the 95% O2/5% CO2 bubbling on. Slices can now be used for electrophysiology or slice treatment.

Data analysis

We prepared acute cerebellar slices of 3 months old mice following the protocol we described here and recorded excitatory postsynaptic potentials (EPSPs) elicited in the Purkinje cell body layer by electrical stimulation of the parallel fibers at various stimulation intensities (Wang et al., 2016). This stimulation elicited a typical P1-N1-P2-N2 waveform (Barnes et al., 2011), where N1 corresponds to the presynaptic fiber volley and N2 to the postsynaptic population spike (Figure 4). Responses elicited by stimulation intensity below 120 mA were too unreliable to be analyzed, and only responses elicited by stimulation intensities above 120 mA were analyzed by calculating the ratio of N2 over N1, which reflects the efficiency of synaptic transmission. At all intensities, the N2/N1 ratio was smaller in calpain-1 KO mice as compared with WT mice, and the overall difference between the two genotypes was statistically significant (Figure 4). The N2/N1 ratios of the EPSPs recorded in cerebellar slices from calpain-1, and PHLPP1 double-KO (DKO) mice at all stimulation intensities were intermediate between those in WT and calpain-1 KO mice, indicating that synaptic transmission was at least partially restored in the cerebellum of DKO mice (Figure 4).


Figure 4. Parallel fibers to Purkinje cell EPSPs are reduced in adult calpain-1 KO mice, and partially restored in adult calpain-1 and PHLPP1 double-KO (DKO) mice. Acute cerebellar slices were prepared as described in Procedure, and field EPSPs were evoked by parallel fiber stimulation recorded in the Purkinje cell layer. Results were calculated as ratios of N2 over N1 and represent means ± SEM of 10 to 11 slices from three to five mice. *P < 0.001, as compared with WT (univariate ANOVA followed by Bonferroni test); #P < 0.001, as compared with WT (univariate ANOVA followed by Bonferroni test); §P < 0.001, as compared with DKO (univariate ANOVA followed by Bonferroni test) (from Wang et al., 2016).

Recipes

  1. Artificial cerebrospinal fluid (aCSF)
    110 mM NaCl
    5 mM KCl
    2.5 mM CaCl2
    1.5 mM MgSO4
    1.24 mM KH2PO4
    10 mM D-glucose
    27.4 mM NaHCO3
  2. Cutting solution
    124 mM NaCl
    26 mM NaHCO3
    10 mM glucose
    3 mM KCl
    1.25 mM KH2PO4
    5 mM MgSO4
    3.4 mM CaCl2
    Cutting solution can be stored at 4 °C for 1 week

Acknowledgments

This work was supported by grant P01NS045260-01 from NINDS (PI: Dr. C.M. Gall) and grant R01NS057128 from NINDS to MB.

Competing interests

There are no conflicts of interest.

Ethics

Animal use in all experiments followed NIH guidelines and all protocols were approved by theInstitution Animal Care and Use Committee of Western University of Health Sciences.

References

  1. Barnes, J. A., Ebner, B. A., Duvick, L. A., Gao, W., Chen, G., Orr, H. T. and Ebner, T. J. (2011). Abnormalities in the climbing fiber-Purkinje cell circuitry contribute to neuronal dysfunction in ATXN1[82Q] mice. J Neurosci 31(36): 12778-12789.
  2. Edwards, F. A., Konnerth, A., Sakmann, B. and Takahashi, T. (1989). A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch 414(5): 600-612.
  3. Kano, M., and Konnerth, A. (1992). Cerebellar slices for patch clamp recording. In: Kettenmann, H. and Grantyn, R. (Eds.). Practical electrophysiological methods. pp: 54-57.
  4. Llano, I., Dreessen, J., Kano, M. and Konnerth, A. (1991a). Intradendritic release of calcium induced by glutamate in cerebellar Purkinje cells. Neuron 7(4): 577-583.
  5. Llano, I., Marty, A., Armstrong, C. M. and Konnerth, A. (1991b). Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices. J Physiol 434: 183-213.
  6. Wang, Y., Hersheson, J., Lopez, D., Hammer, M., Liu, Y., Lee, K. H., Pinto, V., Seinfeld, J., Wiethoff, S., Sun, J., Amouri, R., Hentati, F., Baudry, N., Tran, J., Singleton, A. B., Coutelier, M., Brice, A., Stevanin, G., Durr, A., Bi, X., Houlden, H. and Baudry, M. (2016). Defects in the CAPN1 gene result in alterations in cerebellar development and cerebellar ataxia in mice and humans. Cell Rep 16(1): 79-91.

简介

急性小脑切片广泛用于神经科学家,以研究兴奋性和抑制性突触传递的特性以及涉及其在小脑中调节的细胞内信号传导途径。小脑皮层呈现组织良好的电路,并且可以在急性小脑切片中可靠地刺激和记录若干神经元途径。广泛使用的急性小脑切片制备技术改编自1989年出版的Edwards的薄切片制备方法(Edwards 等人,,1989)。大多数急性小脑切片制备技术使用振动切片机切割来自各种动物物种的新鲜解剖的小脑。在这里,我们介绍一种更简单的方法,它使用组织切碎机快速准备啮齿动物的急性矢状小脑切片。从整个脑中解剖小脑并用组织切碎机切成200-400μm厚的切片。使切片在含氧的aCSF中在37℃下恢复1-2小时。然后切片可用于电生理学或其他类型的实验。该方法可用于从出生后第7至第2年龄的小鼠或大鼠制备小脑切片。该准备比其他方法更快更容易,并提供更多样化的应用程序。
【背景】与许多其他目前的脑切片制备技术一样,急性小脑切片制剂起源于1989年发表的Edwards的薄切片制备方法(Edwards 等人,,1989)。 通常,快速解剖小脑并将其浸入无Ca 2+ + a / aFF并粘合到振动切片机的阶段。 切片后,在使用前将切片在常规aCSF中回收1-2小时(Llano 等人,,1991a和1991b; Kano和Konnerth,1992)。 在这里,我们介绍一种改编自我们实验室使用的急性海马切片制备方法的方法。 新鲜解剖的小脑在McIlwain组织切碎机上快速切片(约10秒),切片过程中无需胶水或氧合作用。 使用该方法制备的切片与用振动切片机制备的切片一样健康,并且可以用于电生理学或各种其他操作。

关键字:小脑快速切片, 小脑, 老鼠, 鼠, 电生理

材料和试剂

  1. 一次性移液器(VWR,目录号:16001-180)
  2. Parafilm(VWR,目录号:52858-000)
  3. 60毫米和100毫米培养皿(VWR,目录号:25373-085和25373-100)
  4. 滤纸(VWR,目录号:28460-030)
  5. 画笔
  6. 气体分散管(Ace Glass,目录号:9435-10)
  7. 老鼠
    1. 成年C57BL / 6(WT)小鼠
    2. 成年钙蛋白酶-1 KO小鼠
    3. 成年钙蛋白酶-1 PHLPP1双KO(DKO)小鼠
  8. 异氟醚
  9. 氯化钠
  10. 氯化钾
  11. CaCl 2
  12. 硫酸镁<子> 4
  13. d葡萄糖
  14. 的NaHCO <子> 3
  15. KH <子> 2 PO <子> 4
  16. 人工脑脊液(aCSF)(见食谱)
  17. 切割解决方案(见食谱)

设备

  1. 寒冷的解剖工具
    1. 角形尖端解剖器剪刀(Fine Science Tools,目录号:14082-09)
    2. 剪刀(精细科学工具,目录号:91401-14)
    3. 弯嘴钳(精细科学工具,目录号:11051-10)
    4. 手术刀片
    5. 解剖刮刀
  2. 脑切片管理器(AutoMate Scientific,目录号:BSK4)
  3. 烧杯
  4. 冰盒&nbsp;
  5. McIlwain Tissue Chopper(Mickle Laboratory Engineering,Brinkmann)
  6. 水浴
  7. 95%O 2 / 5%CO 2 空气罐
  8. -80°C冰柜

程序

  1. 准备1升人工脑脊液(aCSF)(见食谱1)。&nbsp;
  2. 将500毫升新鲜制备的aCSF倒入脑切片容器中。将脑切片容器放入37°C水浴中。将脑切片容器连接到95%O 2 / 5%CO 2 空气罐并给aCSF充氧。气流压力设定在约2磅/平方英寸。
  3. 将100毫升切割溶液倒入150毫升烧杯中。用封口膜覆盖烧杯,并将其置于-80℃冰箱中5-10分钟,直至溶液具有泥浆稠度。
  4. 将冰冷的切割溶液放在大冰盘上。用连接到95%O 2 / 5%CO 2 空气罐的气体分散管鼓泡溶液5分钟。
  5. 将60毫米培养皿的盖子放在冰上。将圆形滤纸放在盖子上,用切割溶液润湿滤纸。这是大脑解剖阶段。
  6. 冷冻解剖工具包括角形尖端解剖剪刀,大剪刀,弯曲尖镊子,手术刀片,勺子,尖端切除的一次性移液器和冰上的两个解剖刮刀。整个解剖设置如图1所示。


    图1.解剖急性小脑切片准备工作。将一个带有切割液,解剖阶段和解剖工具的烧杯放在一个大冰盘上。

  7. 用异氟醚麻醉动物并用适当的工具(例如,剪刀)斩首。立即用解剖工具提取整个大脑,并将其放入冰冷的切割溶液中。此步骤应在1分钟内完成。
  8. 用冰冷的切割溶液冷却大脑1分钟。
  9. 将大脑置于冷解剖阶段。首先,切断前脑,中脑和髓质。然后,切断脑桥以隔离小脑(图2)。


    图2.从小鼠全脑中分离小脑的步骤。切断前脑,中脑和髓质,然后切除小脑下的脑桥。顶部,鼠脑的轴视图。底部,小脑和脑桥的冠状视图。数字和虚线表示切口的步骤和位置。

  10. 立即将小脑转移到McIlwain Tissue Chopper的舞台上。排出组织周围的额外溶液。将小脑切成200-400μm厚的矢状切片(图3)。


    图3. McIlwain Tissue Chopper (Mickle Laboratory Engineering,Brinkmann)

  11. 将小脑移至含有冰冷切割溶液的100毫米培养皿中,小心地将每个切片分开,不要使用两个小刷子损坏它们。
  12. 使用一次性移液管将切片转移到脑切片容器中的温热aCSF中,切割尖端。
  13. 将切片在37℃下在aCSF中孵育1-2小时,其中95%O 2 / 5%CO 2 鼓泡。切片现在可用于电生理学或切片治疗。

数据分析

我们按照我们在此描述的方案制备了3个月大的小鼠的急性小脑切片,并记录了在各种刺激强度下通过电刺激平行纤维在Purkinje细胞体层中引发的兴奋性突触后电位(EPSP)(Wang et al。 ,2016)。这种刺激引发典型的P1-N1-P2-N2波形(Barnes et al。,2011),其中N1对应于突触前纤维截击,N2对应于突触后群体的峰值(图4)。由刺激强度低于120mA引起的响应太不可靠而无法分析,并且仅通过计算N2与N1的比率来分析由120mA以上的刺激强度引起的响应,这反映了突触传递的效率。在所有强度下,与WT小鼠相比,钙蛋白酶-1KO小鼠中的N2 / N1比率更小,并且两种基因型之间的总体差异是统计学显着的(图4)。在所有刺激强度下,来自钙蛋白酶-1和PHLPP1双KO(DKO)小鼠的小脑切片中记录的EPSP的N2 / N1比率介于WT和钙蛋白酶-1KO小鼠之间,表明突触传递至少是在DKO小鼠的小脑中部分恢复(图4)。


图4. Purkinje细胞EPSP的平行纤维在成年钙蛋白-1 KO小鼠中减少,在成年钙蛋白-1和PHLPP1双KO(DKO)小鼠中部分恢复。急性小脑切片制备为在程序中描述,并且通过在浦肯野细胞层中记录的平行纤维刺激诱发野外EPSP。结果计算为N 2与N 1的比率,并且表示来自3至5只小鼠的10至11个切片的平均值±SEM。 * P &lt;与WT相比,0.001(单变量ANOVA,然后是Bonferroni检验); # P &lt;与WT相比,0.001(单变量ANOVA,然后是Bonferroni检验); § P &lt; 0.001,与DKO(单变量ANOVA,然后是Bonferroni检验)相比(来自Wang et al。,2016)。

食谱

  1. 人工脑脊液(aCSF)
    110 mM NaCl
    5 mM KCl
    2.5 mM CaCl 2
    1.5mM MgSO 4
    1.24mM KH 2 PO 4
    10 mM D-葡萄糖
    27.4mM NaHCO 3,
  2. 切割解决方案
    124 mM NaCl
    26 mM NaHCO 3
    10 mM葡萄糖
    3 mM KCl
    1.25mM KH 2 PO 4
    5mM MgSO 4
    3.4 mM CaCl 2
    切割液可以在4°C下储存1周

致谢

这项工作得到NINDS的授权P01NS045260-01(PI:Dr.C.M.Gall)的支持,并授予NINDS的R01NS057128至MB。

利益争夺

没有利益上的冲突。

伦理

所有实验中的动物使用均遵循NIH指南,并且所有方案均获得了批准 西部健康科学大学机构动物护理和使用委员会。

参考

  1. Barnes,J.A.,Ebner,B.A.,Duvick,L.A.,Gao,W.,Chen,G.,Orr,H.T。和Ebner,T.J。(2011)。 攀爬纤维异常 - 浦肯野细胞电路导致 ATXN1神经功能障碍[82Q] 小鼠。 J Neurosci 31(36):12778-12789。
  2. Edwards,F.A.,Konnerth,A.,Sakmann,B。和Takahashi,T。(1989)。 用于哺乳动物中枢神经系统神经元的膜片钳记录的薄片制备。 Pflugers Arch 414(5):600-612。
  3. Kano,M。和Konnerth,A。(1992)。用于膜片钳记录的小脑切片。在:Kettenmann,H。和Grantyn,R。(编辑)。 实用电生理学方法。 pp:54-57。
  4. Llano,I.,Dreessen,J.,Kano,M。和Konnerth,A。(1991a)。 小脑浦肯野细胞谷氨酸诱导钙的Intradendritic释放。 Neuron 7(4):577-583。
  5. Llano,I.,Marty,A.,Armstrong,C。M. and Konnerth,A。(1991b)。 突触和激动剂诱导的大鼠小脑切片中浦肯野细胞的兴奋性电流。 J Physiol 434:183-213。
  6. Wang,Y.,Hersheson,J.,Lopez,D.,Hammer,M.,Liu,Y.,Lee,KH,Pinto,V.,Seinfeld,J.,Wiethoff,S.,Sun,J.,Amouri ,R.,Hentati,F.,Baudry,N.,Tran,J.,Singleton,AB,Coutelier,M.,Brice,A.,Stevanin,G.,Durr,A.,Bi,X.,Houlden, H.和Baudry,M。(2016年)。 CAPN1 基因缺陷导致小脑发育和小脑性共济失调的改变在小鼠和人类中。 Cell Rep 16(1):79-91。
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引用:Wang, Y. and Baudry, M. (2019). Acute Cerebellar Slice Preparation Using a Tissue Chopper. Bio-protocol 9(5): e3187. DOI: 10.21769/BioProtoc.3187.
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