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In vitro Brainstem-spinal Cord Preparation from Newborn Rat
新生大鼠脑干脊髓的体外制备   

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Abstract

The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).

Background

Historically, the in vitro spinal cord of neonatal rodent was developed to study the spinal reflexes (Otsuka and Konishi, 1974). In 1984, Suzue was the first to develop the in vitro brainstem-spinal cord preparation of newborn rat. Thus, it was possible to demonstrate that an isolated central nervous system was able to generate spontaneously what is referred as fictive respiratory activity. Later, it was then possible to determine the location of the CPGs underlying the locomotor rhythm generation (Cazalets et al., 1995; Kjaerulff and Kiehn, 1996; Ballion et al., 2001) and those engaged in respiratory rhythm generation (Smith et al., 1991; Onimaru and Homma, 2003). In our research team, this preparation has been mainly used to study the neural mechanisms underlying the interaction between CPGs. For instance, in a context of interaction between CPGs involved in the same function, our results have contributed to characterize the role played by the sensory afferents and the spinal thoracic segments in the coordination between the cervical and the lumbar locomotor CPGs (Juvin et al., 2005; 2012). Similarly, this preparation allows studies on the neural mechanisms involved in coordination between CPGs engaged in different functions. Based on electrical stimulation of dorsal roots, it was shown that the proprioceptive inputs originating from both hindlimb and forelimb are involved in the respiratory rhythm entrainment observed during locomotion (Morin and Viala, 2002; Giraudin et al., 2012). These ascending entraining signals from the cervical and lumbar afferents are conveyed to the brainstem respiratory centers via a brainstem pontine relay located in the parabrachial/Kölliker-Fuse complex (Giraudin et al., 2012). Using pharmacological and intracellular (patch-clamp recording) approaches on the same preparation, recent results have demonstrated for the first time the existence of an ascending pathway from the lumbar locomotor CPGs to the respiratory CPGs. This central neurogenic mechanism, involving a substance P-dependent modulating mechanism, could play a crucial role in the increased respiratory frequency observed during locomotion (Le Gal et al., 2014). In addition, it was also demonstrated that the locomotor related signal from the lumbar locomotor CPGs selectively modulates the intracellular activity of spinal expiratory neurons (Le Gal et al., 2016). Altogether, our results obtained on the in vitro brainstem spinal cord preparation of new born rat have contributed to increase our understanding of the cellular bases engaged in the coordination of rhythmic neural circuitry responsible for different functions.

Materials and Reagents

  1. Needles (25 G) (Henke-Sass Wolf, catalog number: 4710005016 )
  2. Neonatal rat (0-2 days old)
  3. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
  5. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S0751 )
  6. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  7. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  8. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S8875 )
  9. D-glucose (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC410950010 )
  10. Sodium hydroxide (NaOH) (1 N) (Sigma-Aldrich, catalog number: 795429 )
  11. Hydrochloric acid (HCl) (37%) (Sigma-Aldrich, catalog number: 258148 )
  12. Isoflurane (Piramal Enterprises, Piramal HealthCare, catalog number: Isoflurane )
  13. Vaseline (Sigma-Aldrich, catalog number: 16415 )
  14. Artificial cerebro-spinal fluid (aCSF) solution (see Recipes)

Equipment

  1. FisherbrandTM beaker (1 L) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 15449083 )
  2. Ice bucket (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: M18848-2001 )
  3. Induction chamber for inhalational anesthesia (TemSega, catalog number: chamber )
  4. Isoflurane vaporizer (TemSega, model: Isoflurane vaporizer )
  5. Carbogen, 95% O2, 5% CO2 (The Linde Group, Linde Gaz, model: Carbogen B50 )
  6. Scalpel blade (LCH MEDICAL PRODUCTS, catalog number: SCX23 ) (Figure 1a)
  7. Small scissors (Moria, model: MC26B ) (Figure 1a)
  8. Fine forceps (Fine Science Tools, Dumont, model: 55 ) (Figure 1a)
  9. Dissection chamber (plastic box, 100 ml) with 2 mm layer of silicone elastomer
  10. Binocular microscope (Olympus, model: SZX7 )
  11. Recording chamber (10 ml) composed by a standard petri dish (60 mm) with 2 mm layer of silicone elastomer
  12. Peristaltic pump (Gilson, model: Minipuls® 3 )

Procedure

  1. Artificial cerebro-spinal fluid (aCSF)
    For the preparation, see explanation in the Recipes section below. Once prepared, place a beaker containing aCSF in an ice bucket throughout experience.

  2. Dissection and isolation of the brainstem-spinal cord
    1. Place a neonatal rat (0-2 days old) in an induction chamber for inhalational anesthesia. Animal is anesthetized with 4% isoflurane until the loss of reflex responsiveness to tail pinching (during 7 to 10 min).
    2. Using a scalpel, swiftly euthanize the animal by decapitation (incision must be performed in front of the ears) (Figure 1b). This allows decerebration just rostrally to the fifth cranial nerves.
    3. Remove the skin and muscles covering the back with forceps and scissors (Figure 1c).
    4. Transfer the preparation into a dissection chamber filled with aCSF at 4 °C and insert needle in each fore- and hind-limb to hold the preparation (slightly stretched) with the dorsal surface upwards (Figure 1d).
    5. Under binocular microscope, hold and lift up the skull bones with fine forceps. Using small scissors, cut the skull bones on both sides in order to expose the brainstem (Figure 1e).
    6. Using the same procedure along vertebral column, cut each vertebra on both sides to expose the spinal cord (dorsal laminectomy) (Figure 1f).
    7. Turn the preparation over in order to maintain its ventral surface upwards (Figure 1g).
    8. Hold and lift up the skull bones with fine forceps (Figure 1h) and cut all ventral and dorsal spinal roots leaving as much length as possible. The inset in Figure 1h shows how the skin, muscles and bones are removed to allow laminectomy to be performed. To avoid damage of brainstem and/or spinal cord, it is crucial to never hold nervous tissue with forceps.
    9. Using a spoon, carefully transfer the brainstem-spinal cord with its ventral and dorsal spinal roots still attached in a 10 ml recording chamber containing circulating aCSF (flow rate, 3-5 ml/min adjusted with the peristaltic pump) (Figure 1i).
    10. In order to ensure mechanical stability, the preparation is pinned down with its ventral surface upward by several pins inserted through the meninges surrounding the brainstem and the spinal cord.
    11. To assess the quality of the procedure, motor output activity in spinal ventral roots is recorded with glass suction electrodes filled with aCSF solution (Figure 2a). Signals (burst of action potentials) are amplified (10,000 times) by differential AC amplifiers (low cutoff, 100 Hz; high cutoff, 1 kHz), digitized and acquired via an analogical/digital interface, and finally stored on a computer. The dissection is successful when motor activities are spontaneously generated by the isolated preparation (Figures 2b-2c).


      Figure 1. Illustrations of brainstem-spinal cord dissection. a. Tools used for dissection; b. Neonatal rat (2 days old) and level of incision for decerebration; c. After decerebration, skin and muscles covering the back are removed; d-f. The dorsal surface of the brainstem and the spinal cord is first exposed; g and h. The ventral surface; i. Image showing the isolated brainstem-spinal cord preparation with its spinal roots still attached.


      Figure 2. Motor output activity in spinal ventral roots from in vitro newborn rat preparation. a. Image of an isolated brainstem-spinal cord (from a P2 newborn animal) in a recording chamber. When required, the recording chamber is partitioned either 2 or 3 compartments with barriers of syringe-ejected Vaseline to permit differential exposure of selected spinal cord regions to pharmacological treatment. b. Simultaneous recordings are made from the 4th cervical (C4) and the 1st lumbar (L1) ventral roots. Respiratory-like rhythm is spontaneously generated, showing clear inspiratory (Insp) C4 and pre-inspiratory/expiratory (pre-I; Exp) L1 alternated phases. c. Blind techniques (with sharp or patch electrodes) were used to record rhythmic activity of brainstem and spinal respiratory neurons. Here, the firing of the impaled neuron is in synchrony with L1 ventral root discharge and therefore is identified as an expiratory motoneuron (MN).

Data analysis

  1. Respiratory and locomotor-related activities in spinal ventral roots can be recorded using glass suction electrodes filled with aCSF solution. The motor outputs generated for these two functions consist in rhythmic bursting activities, allowing several type of analysis. Thus, by recording the respiratory motor outputs (see Giraudin et al., 2008), it is possible to determine the respiratory frequency before, during and after pharmacological or lesion experiments. In order to quantify the modulation of the respiratory rhythm between different conditions, statistical analyses are performed using dedicated software, such as SigmaPlot 11.0 (Systat). The results are expressed as mean ± SEM (or SD), and Student’s t-test are used to compare the means of two groups, whereas repeated measures ANOVA and subsequent Tukey’s post hoc tests are used to compare more than two groups. The differences are considered statistically significant when P ˂ 0.05.
  2. It is also possible to establish the phase relationship between motor-related bursting activities, such as respiratory vs. locomotor, or locomotor vs. locomotor bursting activities. In order to calculate the phase relationship, several burst activities of a given ventral root are selected and taken as reference. The phase values of burst onsets in a second ventral root are calculated by dividing the duration between the onsets of two consecutive burst onsets of each motor output by the reference cycle period. The phase values are then plotted on a circular phase diagram with a scale ranging from 0 to 1. A phase value approaching 0 or 1 indicates synchrony, whereas values close to 0.5 reflects alternation.

Recipes

  1. Artificial cerebro-spinal fluid (aCSF) solution
    1. Add 7.305 g NaCl (final concentration 100 mM), 0.249 g KCl (4 mM), 0.069 g NaH2PO4 (1.2 mM), 0.185 g CaCl2 (2 mM), 0.233 g MgCl2 (1.3 mM), 1.764 g NaHCO3 (25 mM), 5.406 g D-glucose (30 mM) in a beaker filled with 1 L of distilled water
    2. Mix the solution with a magnetic stirrer
    3. Place the beaker in an ice bucket and perfuse the aCSF with 95% O2 and 5% CO2 for at least 30 min before use
    4. Adjust the pH to 7.4 (use NaOH or HCl)

Acknowledgments

This protocol was adapted from our published paper: Le Gal et al. (2016), J-P. Le Gal and A. Nicolosi were supported by a doctoral studentship from the French “Ministère de l’Enseignement Supérieur et de la Recherche”. Authors thank R. Anselm for his comments and for English revision.

References

  1. Ballion, B., Morin, D. and Viala, D. (2001). Forelimb locomotor generators and quadrupedal locomotion in the neonatal rat. Eur J Neurosci 14(10): 1727-1738.
  2. Cazalets, J. R., Borde, M. and Clarac, F. (1995). Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat. J Neurosci 15(7 Pt 1): 4943-4951.
  3. Giraudin, A., Cabirol-Pol, M. J., Simmers, J. and Morin, D. (2008). Intercostal and abdominal respiratory motoneurons in the neonatal rat spinal cord: spatiotemporal organization and responses to limb afferent stimulation. J Neurophysiol 99(5): 2626-2640.
  4. Giraudin, A., Le Bon-Jego, M., Cabirol, M. J., Simmers, J. and Morin, D. (2012). Spinal and pontine relay pathways mediating respiratory rhythm entrainment by limb proprioceptive inputs in the neonatal rat. J Neurosci 32(34): 11841-11853.
  5. Juvin, L., Le Gal, J. P., Simmers, J. and Morin, D. (2012). Cervicolumbar coordination in mammalian quadrupedal locomotion: role of spinal thoracic circuitry and limb sensory inputs. J Neurosci 32(3): 953-965.
  6. Juvin, L., Simmers, J. and Morin, D. (2005). Propriospinal circuitry underlying interlimb coordination in mammalian quadrupedal locomotion. J Neurosci 25(25): 6025-6035.
  7. Juvin, L., Simmers, J. and Morin, D. (2007). Locomotor rhythmogenesis in the isolated rat spinal cord: a phase-coupled set of symmetrical flexion extension oscillators. J Physiol 583(Pt 1): 115-128.
  8. Kjaerulff, O. and Kiehn, O. (1996). Distribution of networks generating and coordinating locomotor activity in the neonatal rat spinal cord in vitro: a lesion study. J Neurosci 16(18): 5777-5794.
  9. Le Gal, J. P., Juvin, L., Cardoit, L. and Morin, D. (2016). Bimodal respiratory-locomotor neurons in the neonatal rat spinal cord. J Neurosci 36(3): 926-937.
  10. Le Gal, J. P., Juvin, L., Cardoit, L., Thoby-Brisson, M. and Morin, D. (2014). Remote control of respiratory neural network by spinal locomotor generators. PLoS One 9(2): e89670.
  11. Morin, D. and Viala, D. (2002). Coordinations of locomotor and respiratory rhythms in vitro are critically dependent on hindlimb sensory inputs. J Neurosci 22(11): 4756-4765.
  12. Onimaru, H. and Homma, I. (2003). A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci 23(4): 1478-1486.
  13. Otsuka, M. and Konishi, S. (1974). Electrophysiology of mammalian spinal cord in vitro. Nature 252(5485): 733-734.
  14. Smith, C. J., Ellenberger, H. H., Ballanyi, K., Richter, D. W. and Feldman, J. L. (1991). Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254: 726-729.
  15. Suzue, T. (1984). Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J Physiol 354: 173-183.

简介

新生大鼠的脑干 - 脊髓制备包含能够在没有感觉反馈的情况下产生运动输出的神经网络。 这些神经结构,通常称为中枢模式发生器(CPG),涉及许多重要功能,如呼吸(Morin和Viala,2002; Giraudin等,2008)或运动(Juvin等,2005)。 在这里,我们描述了分离新生大鼠脑干脊髓组织(0-2天)的程序。 双目显微镜下的手术方法允许脑干和脊髓在体外被分离,并且马达输出被记录。 然后,该制剂可用于多种实验方法,例如电生理学,药理学或解剖学研究,并且构成研究CPG之间相互作用的有用模型(Juvin等人,2007; 2012; Giraudin等,2012; Le Gal 等人,2014; 2016)。
【背景】历史上,开发了新生儿啮齿动物的体外脊髓来研究脊柱反射(Otsuka和Konishi,1974)。 1984年,Suzue率先开发出新生大鼠的体外脑脊髓脊髓制剂。因此,有可能证明孤立的中枢神经系统能够自发产生所谓的虚构的呼吸活动。之后,可以确定运动员节奏生成所依赖的CPG的位置(Cazalets等,1995; Kjaerulff和Kiehn,1​​996; Ballion等,2001)和那些从事呼吸节律生成(Smith et al ,1991; Onimaru和Homma,2003)。在我们的研究团队中,该准备主要用于研究CPG之间相互作用的神经机制。例如,在参与相同功能的CPG之间的相互作用的情况下,我们的结果有助于表征感觉传入和脊柱胸段在颈椎和腰椎运动CPG之间协调中的作用(Juvin et al。 ,2005; 2012)。同样,这项准备可以研究参与不同功能的CPG之间协调的神经机制。基于对背根的电刺激,显示源自后肢和前肢的本体感受参与了运动期间观察到的呼吸节律夹带(Morin和Viala,2002; Giraudin等,2012)。来自颈椎和腰椎传入的这些上升夹带信号通过位于parabrachial /Kölliker-Fuse综合体(Giraudin等人,2012)的脑干脑桥继电器传达到脑干呼吸中心。使用相同制剂的药理学和细胞内(膜片钳记录)方法,最近的结果已经证明了首次存在从腰椎运动性CPG到呼吸性CPG的升高途径。这种涉及物质P依赖性调节机制的中枢神经原性机制可能在运动期间观察到的呼吸频率增加中起重要作用(Le Gal等,2014)。此外,还证实,来自腰椎运动员CPG的运动相关信号选择性调节脊髓呼气神经元的细胞内活性(Le Gal等,2016)。总之,我们在新生大鼠的体外脑干脊髓制备中获得的结果有助于增加对从事协调不同功能的节律神经电路的细胞基因的理解。

材料和试剂

  1. 针(25G)(Henke-Sass Wolf,目录号:4710005016)
  2. 新生大鼠(0-2天龄)
  3. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  4. 氯化钾(KCl)(Sigma-Aldrich,目录号:P3911)
  5. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S0751)
  6. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  7. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  8. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S8875)
  9. D-葡萄糖(Thermo Fisher Scientific,Fisher Scientific,目录号:AC410950010)
  10. 氢氧化钠(NaOH)(1N)(Sigma-Aldrich,目录号:795429)
  11. 盐酸(HCl)(37%)(Sigma-Aldrich,目录号:258148)
  12. 异氟烷(Piramal Enterprises,Piramal HealthCare,目录号:异氟烷)
  13. 凡士林(Sigma-Aldrich,目录号:16415)
  14. 人工脑脊液(aCSF)溶液(见Recipes)

设备

  1. 将Fisherbrand TM烧杯(1L)(Thermo Fisher Scientific,Fisher Scientific,目录号:15449083)
  2. 冰桶(SP Scienceware - Bel-Art Products - H-B Instrument,目录号:M18848-2001)
  3. 用于吸入麻醉的感应室(TemSega,目录号:室)
  4. 异氟烷蒸发器(TemSega,型号:异氟烷蒸发器)
  5. Carbogen,95%O 2,5%CO 2(林德集团,Linde Gaz,型号:Carbogen B50)
  6. 手术刀(LCH MEDICAL PRODUCTS,目录号:SCX23)(图1a)
  7. 小剪刀(Moria,型号:MC26B)(图1a)
  8. 精细镊子(Fine Science Tools,Dumont,型号:55)(图1a)
  9. 解剖室(塑料盒,100毫升)与2毫米硅氧烷弹性体层
  10. 双目显微镜(Olympus,型号:SZX7)
  11. 由具有2mm硅氧烷弹性体层的标准培养皿(60mm)组成的记录室(10ml)
  12. 蠕动泵(Gilson,型号:Minipuls 3)

程序

  1. 人工脑脊液(aCSF)
    有关准备,请参阅下面的"配方"部分中的说明。一旦准备好,将含有CSF的烧杯置于冰桶中。

  2. 脑干脊髓的解剖和隔离
    1. 将新生大鼠(0-2天大)在诱导室吸入麻醉。用4%异氟烷麻醉动物,直到对尾巴收缩(在7至10分钟内)的反射反应性丧失。
    2. 使用手术刀,通过断头术迅速安乐死(切口必须在耳朵前面进行)(图1b)。这允许只矫正到第五脑神经
    3. 用镊子和剪刀去除覆盖在背部的皮肤和肌肉(图1c)
    4. 将制剂转移到填充有aCSF在4℃的解剖室和插入针在每个前肢和后肢,以保持准备(轻微拉伸)与背面向上(图1d)。
    5. 在双目显微镜下,用细镊子举起并举起颅骨。使用小剪刀,切两侧的颅骨,以暴露脑干(图1e)。
    6. 沿着脊柱使用相同的程序,切割两侧的每个脊椎暴露脊髓(背椎板切除术)(图1f)。
    7. 转动准备工作,以保持腹侧表面向上(图1g)。
    8. 用细镊子握住并抬起颅骨(图1h),切除所有腹侧和背侧脊髓根,留下尽可能长的长度。图1h中的插图示出了如何去除皮肤,肌肉和骨骼以允许进行椎板切除术。为了避免损伤脑干和/或脊髓,至关重要的是不要用镊子抓住神经组织
    9. 使用勺子,小心地转移脑干脊髓,其腹侧和背侧脊根仍然附着在含有循环aCSF(流速,3-5ml /分钟用蠕动泵调整)的10ml记录室(图1i)。
    10. 为了确保机械稳定性,通过插入通过围绕脑干和脊髓的脑膜的多个销将制备物的腹侧表面向上钉住。
    11. 为了评估该程序的质量,用填充有aCSF溶液的玻璃吸引电极记录脊髓腹根中的运动输出活动(图2a)。通过差分AC放大器(低截止,100Hz;高截止,1kHz)将信号(动作电位突发)放大(10,000次),数字化并通过模拟/数字接口获取,并最终存储在计算机上。当运动活动由孤立制剂自发产生时,解剖是成功的(图2b-2c)

      图1.脑干 - 脊髓剥离的示例。用于解剖的工具; b。新生大鼠(2日龄)和切口水平进行去脑; C。去牙后,去除覆盖背部的皮肤和肌肉; d-f。首先暴露脑干和脊髓的背侧表面; g和h。腹面;一世。图像显示孤立的脑干脊髓准备与其脊根仍然附加

      图2.来自体外新生大鼠准备的脊髓侧根中的运动输出活动。在记录室中的分离的脑干脊髓(来自P2新生动物)的图像。当需要时,记录室被隔开具有注射器喷射凡士林的屏障的2或3个区室,以允许所选脊髓区域的差异暴露于药物治疗。 b。同时记录从第四颈(C4)和第一腰椎(L1)腹侧根。呼吸样节律自发产生,显示清楚的吸气(Insp)C4和预吸气/呼气(pre-I; Exp)L1交替相。 C。盲法(使用尖锐或贴片电极)用于记录脑干和脊髓呼吸神经元的节律活动。这里,刺穿的神经元的发射与L1腹侧根部放电同步,因此被识别为呼气运动神经元(MN)。

数据分析

  1. 可以使用填充有aCSF溶液的玻璃抽吸电极记录脊髓腹根中的呼吸和运动相关活动。为这两个功能产生的电机输出包括节奏爆发活动,允许几种类型的分析。因此,通过记录呼吸运动输出(参见Giraudin等人,2008),可以确定药理学或损伤实验之前,期间和之后的呼吸频率。为了量化不同条件之间的呼吸节律的调节,使用专用软件(例如SigmaPlot 11.0(Systat))进行统计分析。结果表示为平均值±SEM(或SD)和Student's t检验用于比较两组的平均值,而重复测量ANOVA和随后的Tukey氏事后检验用于比较更多比两组。当 P ˂0.05时,差异被认为具有统计意义
  2. 还可以建立运动相关的爆发活动(例如呼吸相对于运动)或运动相对于运动爆发活动之间的相位关系。为了计算相位关系,选择给定腹侧根的几个爆发活动作为参考。通过将每个电动机输出的两个连续突发开始的开始之间的持续时间除以基准周期周期来计算第二腹侧根中的突发开始的相位值。然后将相位值绘制在具有从0到1的刻度范围的圆形相图上。接近0或1的相位值表示同步,而接近0.5的值反映交替。

食谱

  1. 人工脑脊液(aCSF)溶液
    1. 加入7.305g NaCl(终浓度100mM),0.249g KCl(4mM),0.069g NaH 2 PO 4(1.2mM),0.185g CaCl 2, 2mM(2mM),0.233g MgCl 2(1.3mM),1.764g NaHCO 3(25mM),5.406g D-葡萄糖(30mM) )装入装有1L蒸馏水的烧杯中
    2. 用磁力搅拌器混合溶液
    3. 将烧杯置于冰桶中,在使用前用95%O 2和5%CO 2灌注aCSF至少30分钟
    4. 调节pH至7.4(使用NaOH或HCl)

致谢

该方案改编自我们发表的文章:Le Gal等人。 (2016),J-P。 Le Gal和A. Nicolosi得到了法国"Ministèrede l'EnseignementSupérieuret de la Recherche"博士生的支持。作者感谢R. Anselm的意见和英语修订。

参考文献

  1. Ballion,B.,Morin,D。和Viala,D。(2001)。  前肢运动发生器和新生大鼠的四肢运动。 Eur J Neurosci 14(10):1727-1738。
  2. Cazalets,JR,Borde,M.和Clarac,F。(1995)。  用于新生大鼠后肢运动的中心模式发生器的定位和组织。 15(7 Pt 1):4943-4951。
  3. Giraudin,A.,Cabirol-Pol,MJ,Simmers,J.and Morin,D.(2008)。  在新生大鼠脊髓中的肋间和腹部呼吸运动神经元:时空组织和对肢体传入刺激的反应。神经生理学99 :2626-2640。
  4. Giraudin,A.,Le Bon-Jego,M.,Cabirol,MJ,Simmers,J.and Morin,D。(2012)。  脊髓和脑桥中继途径介导新生大鼠肢体本体感觉输入引起的呼吸节律夹带。 (34):11841-11853。
  5. Juvin,L.,Le Gal,JP,Simmers,J.和Morin,D。(2012)。  哺乳动物四肢运动中的宫颈腰椎协调:脊柱胸廓回路和肢体感觉输入的作用。 32(3):953-965。
  6. Juvin,L.,Simmers,J.和Morin,D.(2005)。  Propriospinal电路底层interlimb协调在哺乳动物四肢运动。  J Neurosci 25(25):6025-6035。
  7. Juvin,L.,Simmers,J.和Morin,D.(2007)。 
  8. Kjaerulff,O.和Kiehn,O。(1996)。  在新生大鼠脊髓中产生和协调运动活性的网络的分布::病变研究。 16 Neurorosci 16(18):5777-5794 。
  9. Le Gal,JP,Juvin,L.,Cardoit,L.和Morin,D.(2016)。  新生大鼠脊髓中的双模呼吸运动神经元。 J Neurosci 36(3):926-937。
  10. Le Gal,JP,Juvin,L.,Cardoit,L.,Thoby-Brisson,M.and Morin,D.(2014)。  通过脊髓运动发生器远程控制呼吸神经网络。 9(2):e89670。 br />
  11. Morin,D。和Viala,D。(2002)。  运动和呼吸节律的协调在体外严重依赖于后肢感觉输入。 J Neurosci 22(11):4756-4765。
  12. Onimaru,H。和Homma,I。(2003)。  23(4):1478-1486。
  13. Otsuka,M.和Konishi,S。(1974)。  哺乳动物脊髓的电生理学。 252(5485):733-734。
  14. Smith,CJ,Ellenberger,HH,Ballanyi,K.,Richter,DWand Feldman,JL(1991)。  Pre-Bötzingercomplex:可能在哺乳动物中产生呼吸节律的脑干区域 254:726-729。
  15. Suzue,T。(1984)。  呼吸节律生成在新生大鼠的体外脑干 - 脊髓制备中。 354:173-183。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Le Gal, J., Nicolosi, A., Juvin, L. and Morin, D. (2016). In vitro Brainstem-spinal Cord Preparation from Newborn Rat. Bio-protocol 6(22): e2003. DOI: 10.21769/BioProtoc.2003.
  2. Le Gal, J. P., Juvin, L., Cardoit, L. and Morin, D. (2016). Bimodal respiratory-locomotor neurons in the neonatal rat spinal cord. J Neurosci 36(3): 926-937.
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