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Transpharyngeal Exposure of GnRH Neurons
GnRH神经元的前路寰枢暴露研究

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Abstract

The neurons secreting gonadotropin-releasing hormone (GnRH) control fertility in all mammalian species. However, investigations of this neuronal population are difficult as the cell bodies of the approximately 800 GnRH neurons are scattered through the basal forebrain ranging from the olfactory bulbs through to the base of the hypothalamus. While acute brain slice preparations enable the electrophysiological characteristics of these cells to be determined in vitro, their topography has made investigations in vivo extremely difficult. We detail here a surgical approach that allows GnRH neurons at the base of the hypothalamus to be assessed in vivo in the anesthetized mouse (Figure 1). This procedure enables electrical recordings to be made from neurons located on the ventral surface of the mouse brain (Constantin et al., 2013).

Keywords: GnRH neurons(下丘脑促性腺激素释放激素神经元), In vivo(在体内), Electrophysiology(电生理学)



Figure 1. Exposure of AHA-GnRH neurons

Materials and Reagents

  1. GnRH-GFP mice (Spergel et al., 1999; Suter et al., 2000)
  2. Pentobarbital
  3. CaCl2
  4. MgCl2
  5. HEPES
  6. NaHCO3
  7. D-glucose
  8. Sucrose
  9. O2 Medical Grade
  10. 5% CO2 Carbogen
  11. Artificial cerebrospinal fluid (aCSF) (see Recipes)

Equipment

  1. Set Up
    1. Metal plate 200 x 200 x 1 mm
    2. Binocular microscope (Leica Microsystems, model: M80 )
    3. Cold light source (Leica Microsystems, model: CLS 150X )
    4. Upright microscope (OLYMPUS, model: BX51 )
    5. Long working distance objective (20x M Plan Apo NA 0.42) (Mitutuyo, catalog number: 378-804-2 )
    6. Blue fluorescence illumination (OLYMPUS, model: U-MGGFPHQ ) (excitation wavelength centered at 470 nm with high pass emission filtered at 517 nm)
    7. AC/DC bioamplifier (CWE, model: BMA-200 )
    8. Analogic-to-digital converter (Molecular Devices, model: Digidata 1332A )
    9. Subcutaneous electrodes (platinum ~20 G) hooked to the bioamplifer
    10. Ventilator (CWE, model: SAR-830/P) with the following parameters: 100 breaths/min, with constant pressured cycles set at 1.4 cm H2O, 0.3 s/inspiration, O2 90–80 ml/min
    11. Body temperature controller (CWE, model: CT-1000) with heat pad and rectal probe, set at 37 °C

  2. Surgery
    1. Stainless steel blocks (L x W x H: 60 x 40 x 10 mm)
      1. Home-made block, inclinable with 2 screw = “pillow” (Figure 2)


        Figure 2. Pillow

      2. Home-made block, inclinable with 2 screw and holding a stainless steel spatula narrowed down to 3 mm wide with a fine file to fit in the mouth of a mouse, bent into hook (about 0.80 cm diameter) = central separator (Figure 3)


        Figure 3. Central separator

    2. Syringe (26 G needle) for intraperitoneal and intramuscular injections
    3. Syringe (23 G needle) for chilled aCSF
    4. Forceps Dumont No 5
    5. Small scissors
    6. Spring scissors 8 mm blades (Fine Science Tools, catalog number: 15024-10)
    7. Bone scissors Bone Cutting Spring Scissors (Fine Science Tools, catalog number: 16144-13)
    8. Agricola retractor 3.5 cm spread (Fine Science Tools, catalog number: 17005-04)
    9. Micro-drill (GEBR Brasseler; Ideal Micro-Drill) (PW Stoelting, catalog number: 58610)
    10. Round burr tip (o.d. 0.5 mm)
    11. Bone wax (Johnson & Johnson, Ethicon, model: W810)
    12. Syringe body holding a 23 G needle taped into a hook = needle hook
    13. Dental vacuum connected with 1-ml syringe body holding a cut 23 G needle (23 G-needle dental vacuum) or 1-ml insulin syringe holding a cut 30 G needle (30 G-needle dental vacuum)
    14. 50-ml syringe for inflow (gravity) with 1 home-made magnetic holder
    15. Peristaltic pump for outflow with a cut 23 G needle held by home-made magnetic holder (Figure 4)


      Figure 4. Magnetic holder

    16. Cotton balls
    17. Tissue paper, separated before use
    18. Lab tape
    19. Cotton sewing thread No 50
    20. Super glue with brush
    21. Polyethylene catheter (o.d. 0.96 x i.d. 0.58 mm)
    22. Lighter

Software

  1. ClampEx pCLAMP 10 Electrophysiology Data Acquisition and Analysis Software

Procedure

Note: Build the apparatus on a mobile metal plate so that the whole apparatus can be moved from the binocular microscope to the upright microscope.

  1. Make 1 liter of aCSF and oxygenate it with 5% CO2 carbogen.
  2. Fill 1 ml syringe with aCSF and keep it on ice (= chilled aCSF).
  3. Turn on the heat pad under the binocular scope.
  4. Prepare the ventilation catheter.
    1. Cut to about 6 cm long.
    2. Bend it with a lighter in the middle at 90 degrees.
    3. Cut it with an angle (45 degrees) at about 0.4 cm from the middle bend.
    4. Attach it to the ventilator tubing.
  5. Prepare a piece of cotton thread, 10 cm long.
  6. Prepare another piece of cotton thread, 10 cm long, and make a loop with it.
  7. Prepare pieces of tape for paws (about 3 cm long each).
  8. Inject the mouse intraperitoneally (IP) with pentobarbital [PB (10 mg/ml); 0.4 mg/10 g body weight].
  9. Place it as soon as possible on the heat pad (pentobarbital evokes hypothermia); and secure it with a piece of tape across the tail as it becomes fully anesthetized.
  10. Place the mouse on its back, its head on one of the stainless steel blocks.
    Note: For a right-handed experimenter, the head is on the left, the tail on the right for an optimal drilling angle.
  11. Place the rectal probe and secure it with a piece of tape with the tail on the pad.
  12. Tape the front paws on the block (view in Figure 6 at the end).
  13. Add PB intramuscularly (10 mg/ml) (0.05 ml of each leg).
  14. Place the looped thread behind the front teeth.
  15. Use the second block to pull the thread back and maintain the neck extended; the trachea should bulge a little.
  16. Check the mouse is deeply anesthetized by toe pinching.
  17. Use the suprasternal notch as an inferior boundary and the chin as a superior boundary for the initial incision.
  18. Make a hole in the skin above the suprasternal notch with the small scissors.
  19. Continue the incision along the midline to the chin but avoid cutting the pink (non-furry) skin of the chin itself as this bleeds.
  20. Cut the skin laterally on each side, above the suprasternal notch and towards the cheeks (see Figure 5).


    Figure 5. Incisions

  21. Place the subcutaneous ECG electrodes in the right arm and the left leg (lead II) to monitor the heart rate.
  22. Tape all the cables to the metal plate.
  23. Separate the salivary glands with the 2 pairs of forceps and move them to either side to expose the muscles overlying the trachea.
  24. Hold the muscles and dilacerate them above the sternum.
  25. Lift them progressively until reaching the hyoid bone where they can then be cut off.
  26. Expose and hold the conjunctive tissue above the trachea, lift it gently with one pair of forceps and pass the second pair (hold it closed) beneath.
  27. When the forceps tip has passed behind the trachea, separate the tips gently to detach the trachea underneath.
    Note: Do not lose the sight of the forceps tips because the carotids are very close.
  28. Take the second cotton thread, hold it with the forceps tips and pull it back underneath the trachea.
  29. Prepare a first knot.
  30. Make a hole in the trachea above the first or the second cartilage ring above the sternum with the micro scissors.
  31. Insert the catheter, secure it with the knot.
  32. Tape securely the ventilator tubing on the plate.
  33. Start the ventilator and adjust catheter until the breathing is regular.
  34. Put some glue on the knot, the area the catheter enters in the trachea and on the beginning of the threads to have them well secured.
  35. Cut the threads above the glue to avoid snagging them on the drill later.
  36. Remove the thread from the teeth, tilt the head towards the body, pinch the skin above the scalp with the forceps and make a small incision.
  37. Open the skin to expose the base of the skull, put some glue on the skull and use the jaws to press the head into the stainless steel “pillow” for few seconds.
    Note: Wiggle the teeth to make sure the head is not moving as loose subcutaneous conjunctive tissue can remain.
  38. Detach the thyroid glands from the trachea and flip each of them on the side.
  39. Cut the trachea above the catheter and as it retracts, the esophagus becomes visible.
  40. Hold the esophagus with the forceps, lift it and cut it.
  41. Lift the trachea and the esophagus together and cut them out with the larynx using the micro scissor.
  42. Cut the digastric muscles with the micro scissor at the level of the tendons on both sides and lift them up towards the lips.
    Note: Cut as superficially as possible otherwise it will bleed.
  43. Cut the hyoid bone with the bone scissors down the middle and place the central separator at the back of the tongue.
    Note: This cut is likely to bleed, be prepared to place the retractor very fast; it will apply pressure and help stop bleeding almost immediately.
  44. Cut the soft palate to expose the nasal cavity with the micro scissor.
  45. Make a tiny cotton bud on the tip of a pair of forceps and rub the roof of the nasal cavity to expose the sphenoid bone.
  46. Place the Agricola retractor.
    Note: It is likely to touch blood vessels as it opens, do not try to move it around otherwise it will bleed.
  47. Identify the anatomical landmarks: rostrally, the transverse sinus of the sphenoid bone (dark blue about 0.8 mm wide, transversal to the nasal cavity) and caudally, the bone junction (thin white line, transversal to the nasal cavity, at the end of it).
  48. Make a bigger cotton bud on the forceps and on one side, break the internal pterygoid process by applying a constant pressure and rolling motion towards the bone (the right side, closest to the experimenter gives a better access).
    Note: Control this motion carefully as the bone may break suddenly and  cause major bleeding; minor bleeding is still possible without a sudden break, check it slowly before releasing the pressure of the cotton bud.
  49. Prepare about 10 balls of bone wax (about 0.5 mm diameter) and pick up the first one with the forceps.
  50. Start drilling caudally to the transverse sinus of the sphenoid bone and move very slowly rostrally.
  51. Drill superficially, not more than the depth of the drill head, and only drill horizontally.
  52. Stop as often as possible using the size of one drill head as a measure of progress, add one ball of bone wax and pack it with a mini cotton bud to infiltrate the bone layers. Add more balls of bone wax as needed until the sinus is well clogged.
  53. Continue drilling rostrally using the hard palate above as a landmark.
  54. Start drilling caudally stopping before the bone junction.
  55. Start drilling vertically and slowly to thin the bone until it becomes transparent.
    Note: The red microvasculature on the white optic chiasm is visible through the thinned bone, the sides are lined by the posterior communicating arteries, bulging further on the right side.
  56. Wash thoroughly the whole surgical area with aCSF and the 23 G-needle dental vacuum before removing the thinned bone and exposing the brain.
  57. Make sure to remove all remaining wax, bone dust and fur that could contaminate the exposed brain area.
  58. Grip the side on the thinned bone with the needle hook, lift it up to break it and remove the bone fragment.
  59. Repeat these steps until the optic chiasm is exposed and the very end of the optic tracts are visible.
    Note: Keep the hook pointing towards the midline to avoid touching the posterior communicating arteries; do not hook anything on the front edge of the cavity as the posterior communicating arteries join at the front to circle the optic chiasm.
  60. Inject chilled aCSF under the meninges wrapping the optic chiasm to stiffen both the meninges and the optic chiasm, and fill the whole cavity with the remaining chilled aCSF.
  61. Bring the 23 G-needle dental vacuum vertically and perpendicularly into the hole and suck out the optic chiasm at the center, lift it and progressively tear apart the meninges that are pulled with it on both sides and caudally with the tip of the #5 forceps.
  62. Progress rostrally towards the optic tracts being pulled; pinch them to cut them.
  63. Switch to a 30 G-needle dental vacuum to take the remaining meninges; the exposed cavity is about 2.0 mm long and 0.8 mm wide.
  64. Place the aCSF inflow through a catheter, up in the surgical area (home-made magnetic holder on the plate) to avoid drops.
  65. Place the 23 G-needle outflow as close as possible from the surface of the brain in the rostral side of the exposed area (home-made magnetic holder on the stainless steel “pillow”).
  66. Move the apparatus under the microscope and visualize the GnRH neurons under blue fluorescence.


    Figure 6. Apparatus

Recipes

  1. Artificial cerebrospinal fluid (aCSF)
    118 mM NaCl
    3 mM KCl
    2.5 mM CaCl2
    1.2 mM MgCl2
    10 mM HEPES
    25 mM NaHCO3
    5.5 mM D-glucose
    2.9 mM sucrose
    pH 7.3

Acknowledgments

This protocol is adapted from the article Constantin et al. (2013).

References

  1. Constantin, S., Iremonger, K. J. and Herbison, A. E. (2013). In vivo recordings of GnRH neuron firing reveal heterogeneity and dependence upon GABAA receptor signaling. J Neurosci 33(22): 9394-9401.
  2. Spergel, D. J., Kruth, U., Hanley, D. F., Sprengel, R. and Seeburg, P. H. (1999). GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice. J Neurosci 19(6): 2037-2050.
  3. Suter, K. J., Song, W. J., Sampson, T. L., Wuarin, J. P., Saunders, J. T., Dudek, F. E. and Moenter, S. M. (2000). Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology. Endocrinol 141(1): 412-419.

简介

分泌促性腺激素释放激素(GnRH)的神经元控制所有哺乳动物物种的生育力。 然而,对于这种神经元群体的研究是困难的,因为大约800个GnRH神经元的细胞体通过从嗅球到下丘脑的基底的基底前脑分散。 虽然急性脑切片制剂使得这些细胞的电生理特征在体外得以确定,但它们的形态使得体内的研究非常困难。 我们在这里详细介绍一种手术方法,允许在麻醉小鼠体内评估下丘脑基部的GnRH神经元(图1)。 该过程使得能够从位于小鼠脑的腹侧表面上的神经元进行电记录(Constantin等人,2013)。

关键字:下丘脑促性腺激素释放激素神经元, 在体内, 电生理学



图1. AHA-GnRH神经元的暴露

材料和试剂

  1. GnRH-GFP小鼠(Spergel等人,1999; Suter等人,2000)
  2. 戊巴比妥
  3. CaCl <2>
  4. MgCl 2
  5. HEPES
  6. NaHCO 3
  7. D-葡萄糖
  8. 蔗糖
  9. O 2 医疗级
  10. 5%CO 2 Carbogen
  11. 人工脑脊液(aCSF)(参见配方)

设备

  1. 建立
    1. 金属板200 x 200 x 1 mm
    2. 双目显微镜(Leica Microsystems,型号:M80)
    3. 冷光源(Leica Microsystems,型号:CLS 150X)
    4. 立式显微镜(OLYMPUS,型号:BX51)
    5. 长工作距离物镜(20x M Plan Apo NA 0.42)(Mitutuyo,目录号:378-804-2)
    6. 蓝色荧光照明(OLYMPUS,型号:U-MGGFPHQ)(激发波长集中在470nm,在517nm过滤高通发射)
    7. AC/DC生物放大器(CWE,型号:BMA-200)
    8. 模拟 - 数字转换器(Molecular Devices,型号:Digidata 1332A)
    9. 钩住生物放大器的皮下电极(铂〜20G)
    10. 通风器(CWE,型号:SAR-830/P),具有以下参数:100次呼吸/分钟,恒定压力循环设定在1.4cm H 2 O,0.3s /吸气, 2 <90-80ml/min
    11. 体温控制器(CWE,型号:CT-1000),带有加热垫和直肠探头,设置为37℃

  2. 手术
    1. 不锈钢块(L×W×H:60×40×10mm)
      1. 自制块,可用2个螺丝="枕头"倾斜(图2)


        图2.枕头

      2. 自制块,可用2个螺丝倾斜,并保持一个不锈钢刮刀缩小到3毫米宽与一个精细的文件,以适应鼠标的嘴,弯曲成钩(约0.80厘米直径)=中央分离器(图3)


        图3.中央分隔符

    2. 注射器(26 G针)腹膜内和肌内注射
    3. 冷冻aCSF的注射器(23 G针)
    4. Forceps Dumont No 5
    5. 小剪刀
    6. 弹簧剪刀8mm刀片(Fine Science Tools,目录号:15024-10)
    7. 骨剪刀骨切割弹簧剪刀(Fine Science Tools,目录号:16144-13)
    8. Agricola牵开器3.5厘米(Fine Science Tools,目录号:17005-04)
    9. 微钻(GEBR Brasseler;理想微型钻头)(PW Stoelting,目录号:58610)
    10. 圆形刀头(外径0.5mm)
    11. 骨蜡(Johnson& Johnson,Ethicon,型号:W810)
    12. 注射器主体握住23 G针刺入钩子=针钩
    13. 与保持切口23G针(23G针牙科真空)或1-ml胰岛素注射器,保持切口30G针(30G针牙齿真空)的1ml注射器主体连接的牙科真空。
    14. 50毫升注射器用于流入(重力)与1个自制磁性支架
    15. 蠕动泵用于流出,具有由家用磁性支架(图4)保持的切口23G针

      图4.磁性支架

    16. 棉球
    17. 薄纸,使用前分开,
    18. Lab磁带
    19. 棉缝线50号,
    20. 超级胶水用刷
    21. 聚乙烯导管(o.d. 0.96 x i.d. 0.58mm)
    22. 打火机

软件

  1. ClampEx pCLAMP 10电生理数据采集和分析软件

程序

注意:在移动金属板上构建设备,以便整个设备可以从双目显微镜移动到直立显微镜。

  1. 制备1升aCSF并用5%CO 2碳源使其氧化。
  2. 填充1毫升注射器与aCSF,并保持在冰(=冷冻aCSF)。
  3. 打开双目镜下的加热垫。
  4. 准备通气导管。
    1. 切成约6厘米长。
    2. 用90度的中间打火机弯曲。
    3. 从中间弯曲处以约0.4厘米的角度(45度)切割。
    4. 将其连接到呼吸机管。
  5. 准备一条10厘米长的棉线。
  6. 准备另一条棉线,10厘米长,并与它做一个环
  7. 准备爪子的磁带(每个约3厘米长)。
  8. 用戊巴比妥[PB(10mg/ml)]腹腔注射(IP)小鼠; 0.4mg/10g体重]
  9. 将其尽快放在热垫上(戊巴比妥引起低体温); 并用一条胶带将其固定在尾部,因为它完全麻醉
  10. 将鼠标放在它的背部,它的头部在一个不锈钢块。
    注意:对于右手的实验者,头部在左侧,尾部在右侧,以获得最佳钻削角度。
  11. 放置直肠探头,并用一块胶带将尾巴固定在衬垫上。
  12. 将前爪贴在块上(图6中的视图结束)。
  13. 肌肉内加入PB(10mg/ml)(每条腿0.05ml)
  14. 将环形线穿过前牙。
  15. 使用第二块拉回线并保持颈部伸展; 气管应凸起一点。
  16. 检查鼠标是否由于趾缩而深度麻醉。
  17. 使用suprasternal凹口作为下边界和下巴作为上边界的初始切口
  18. 使用小剪刀在上部切口上方的皮肤上打孔。
  19. 肌肉内加入PB(10mg/ml)(每条腿0.05ml)
  20. 将环形线穿过前牙。
  21. 使用第二块拉回线并保持颈部伸展; 气管应凸起一点。
  22. 检查鼠标是否由于趾缩而深度麻醉。
  23. 使用suprasternal凹口作为下边界和下巴作为上边界的初始切口
  24. 使用小剪刀在上部切口上方的皮肤上打孔。
  25. ... Hold the muscles and dilacerate them above the sternum.
  26. Lift them progressively until reaching the hyoid bone where they can then be cut off.
  27. Expose and hold the conjunctive tissue above the trachea, lift it gently with one pair of forceps and pass the second pair (hold it closed) beneath.
  28. When the forceps tip has passed behind the trachea, separate the tips gently to detach the trachea underneath.
    Note: Do not lose the sight of the forceps tips because the carotids are very close.
  29. Take the second cotton thread, hold it with the forceps tips and pull it back underneath the trachea.
  30. Prepare a first knot.
  31. Make a hole in the trachea above the first or the second cartilage ring above the sternum with the micro scissors.
  32. Insert the catheter, secure it with the knot.
  33. Tape securely the ventilator tubing on the plate.
  34. Start the ventilator and adjust catheter until the breathing is regular.
  35. Put some glue on the knot, the area the catheter enters in the trachea and on the beginning of the threads to have them well secured.
  36. Cut the threads above the glue to avoid snagging them on the drill later.
  37. Remove the thread from the teeth, tilt the head towards the body, pinch the skin above the scalp with the forceps and make a small incision.
  38. 打开皮肤暴露颅骨的基部,在颅骨上涂一些胶水,并使用颚子将头部进入不锈钢"枕头"几秒钟。
    注意:摇晃牙齿以确保头部不会因为皮下松动而移动。
  39. 从气管分离甲状腺,并在侧面翻转他们每一个
  40. 切开导管上方的气管,收缩时,食道变得可见
  41. 用钳子握住食道,举起并切开它
  42. 将气管和食管一起抬起,用微型剪刀将喉头切开。
  43. 用微型剪刀在两侧的肌腱水平处切开双腹肌,并将其向嘴唇提起。
    注意:尽可能简洁地切开,否则会出血。
  44. 用中间的骨剪刀剪下舌骨,将中央分离器放在舌头的后面 注意:这切口很可能出血,准备好非常快地放置牵开器;它会 施加压力,并帮助几乎立即停止出血。
  45. 切开软腭,用微型剪刀暴露鼻腔
  46. 在一副镊子的尖端做一个小棉花芽,擦鼻腔的屋顶暴露蝶窦骨。
  47. 放置Agricola牵开器。
    注意:打开血管时可能会触摸血管,不要试图移动血管,否则会出血。
  48. 识别解剖标志:rostrally,蝶骨的横向窦(深蓝色约0.8 mm宽,横向对鼻腔)和尾部,骨连接点(稀白线,横向对鼻腔,在末端)。
  49. 在镊子和一侧做一个更大的棉花芽,通过施加一个恒定的压力和向骨头(右侧,最接近实验者提供更好的访问)打破内部翼状突过程。
    注意:小心控制此运动,因为骨头可能突然断裂, 轻微出血仍然可能没有突然中断,在释放棉花芽的压力前慢慢检查它。
  50. 准备约10个骨蜡(直径约0.5毫米)球,并拿起第一个用镊子
  51. 开始钻向蝶窦骨的横窦,并向右侧移动非常缓慢
  52. 钻头表面,不大于钻头的深度,只能水平钻孔
  53. 停止尽可能多的使用一个钻头的大小作为进度的测量,添加一个骨蜡的球和包装它与一个迷你棉花芽渗透骨层。根据需要添加更多的骨蜡球,直到窦良好堵塞。
  54. 继续使用上面的硬腭作为地标来钻孔。
  55. 开始在骨接合点之前在尾部钻孔停止。
  56. 开始垂直和缓慢钻取薄骨,直到它变得透明。
    注意:白色视交叉上的红色微血管通过变薄的骨骼可见,两侧由后交通动脉排列,在右侧进一步凸起。
  57. 用aCSF和23 G针牙真空彻底清洗整个手术区域,然后取出变薄的骨头并暴露脑。
  58. 确保清除可能污染暴露的大脑区域的所有残留的蜡,骨灰和毛皮
  59. 用针钩抓住变薄的骨骼上的一侧,将其提起以打破骨骼并移除骨碎片
  60. 重复这些步骤,直到光学交联曝光,并且光束的最后端可见 注意:保持钩子指向中线,以避免接触后交通动脉;不要在腔前边缘上挂钩任何东西,因为后面的连通动脉在前面连接以围绕视交叉。
  61. 将冷冻的aCSF注入包裹视交叉的脑膜下,以加固脑膜和视交叉,并用剩余的冷冻aCSF填充整个空腔。
  62. 将23 G针牙科真空垂直和垂直地进入孔中,吸出中心的视交叉,提起它,并逐步撕裂两侧和尾部与#5镊子的尖端牵引的脑膜。
  63. 朝向被拉的视神经前进;捏他们剪它们
  64. 切换到30 G针牙科真空吸取剩余的脑膜;暴露的空腔长约2.0毫米,宽0.8毫米
  65. 通过导管将aCSF流入,放置在手术区域(板上的自制磁性支架),以避免液滴。
  66. 将23 G针流出尽可能靠近暴露区域(不锈钢"枕头"上的自制磁性支架)的头侧的大脑表面。
  67. 在显微镜下移动设备,并在蓝色荧光下可视化GnRH神经元

    图6.装置

食谱

  1. 人工脑脊液(aCSF)
    118 mM NaCl
    3 mM KCl
    2.5mM CaCl 2·h/v 1.2mM MgCl 2·h/v 10 mM HEPES
    25mM NaHCO 3/v/v 5.5mM D-葡萄糖 2.9mM蔗糖 pH 7.3

致谢

该协议改编自Constantin等人(2013)的文章。

参考文献

  1. Constantin,S.,Iremonger,K.J.and Herbison,A.E。(2013)。 记录GnRH神经元放电显示GABA A受体的异质性和依赖性 J Neurosci 33(22):9394-9401。
  2. Spergel,D.J.,Kruth,U.,Hanley,D.F.,Sprengel,R。和Seeburg,P.H。(1999)。 转基因小鼠中绿色荧光蛋白标记的促性腺激素释放激素神经元中的GABA和谷氨酸活化通道。 J Neurosci 19(6):2037-2050。
  3. Suter,K.J.,Song,W.J.,Sampson,T.L.,Wuarin,J.P.,Saunders,J.T.,Dudek,F.E.and Moenter,S.M。(2000)。 绿色荧光蛋白对促性腺激素释放激素神经元的遗传靶向:全细胞电生理特性的表征和形态学。内分泌学  141(1):412-419。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Constantin, S. and Herbison, A. E. (2014). Transpharyngeal Exposure of GnRH Neurons. Bio-protocol 4(6): e1080. DOI: 10.21769/BioProtoc.1080.
  2. Constantin, S., Iremonger, K. J. and Herbison, A. E. (2013). In vivo recordings of GnRH neuron firing reveal heterogeneity and dependence upon GABAA receptor signaling. J Neurosci 33(22): 9394-9401.
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