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Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP), a GABAA receptor δ-subunit specific agonist, when present at low (μM) concentrations, preferentially binds and activates extrasynaptic (non-γ2, δ-subunit-containing) GABAARs (Storustovu and Ebert, 2006; Richardson et al., 2011, 2013).
In this prototype saturation binding experiment, a series of concentrations of [3H]gaboxadol (5, 10, 25, 50, 75, 100, 250 and 400 nM) will be used. GABA at 200 μM will be added into binding mixtures as a cold displacer for [3H]gaboxadol. Slide mailers are used and each requires 7 ml binding mixture. Pre-, post-washing and binding buffer is 50 mM Tris-Citrate (pH 7.1). The detailed procedure is outlined below.

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Autoradiographic 3H-Gaboxadol Receptor Binding Protocol
放射自显影3H-Gaboxadol受体结合法

神经科学 > 细胞机理 > 受体-配体结合
作者: Lynne Ling
Lynne LingAffiliation: Department of Pharmacology, School of Medicine, Southern Illinois University, Springfield, IL, USA
Bio-protocol author page: a1042
 and Donald Caspary
Donald CasparyAffiliation: Department of Pharmacology, School of Medicine, Southern Illinois University, Springfield, IL, USA
For correspondence: dcaspary@siumed.edu
Bio-protocol author page: a1043
Vol 3, Iss 23, 12/5/2013, 2296 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.989

[Abstract] Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP), a GABAA receptor δ-subunit specific agonist, when present at low (μM) concentrations, preferentially binds and activates extrasynaptic (non-γ2, δ-subunit-containing) GABAARs (Storustovu and Ebert, 2006; Richardson et al., 2011, 2013).
In this prototype saturation binding experiment, a series of concentrations of [3H]gaboxadol (5, 10, 25, 50, 75, 100, 250 and 400 nM) will be used. GABA at 200 μM will be added into binding mixtures as a cold displacer for [3H]gaboxadol. Slide mailers are used and each requires 7 ml binding mixture. Pre-, post-washing and binding buffer is 50 mM Tris-Citrate (pH 7.1). The detailed procedure is outlined below.

[Abstract]

Materials and Reagents

  1. Ice
  2. [3H]gaboxadol (Gift from Merck & Co.)
  3. GABA (Sigma-Aldrich, catalog number: A2129 )
  4. Incubation buffer: 50 mM Tris-Citrate buffer (pH 7.1)
  5. 0.1 M PBS (Phosphate buffer saline, pH 7.4) Binding mixture (see Recipes)

Equipment

  1. Slide mailer (Fisher Scientific, model: HS15986 )
  2. Labeled foil
  3. Cryostat (Leica Microsystems, model: CM1850 )
  4. -20 ℃ freezer
  5. Scintillation counter
  6. Ruler
  7. Diamond scribe
  8. Double-sided tape
  9. Phosphor transcreens
  10. Cyclone Phosphor Imager (PerkinElmer)

Software

  1. OptiQuant Version 4.0 (PerkinElmer)
  2. GraphPad Radioactive Calculator (GraphPad Software)

Procedure

  1. Animal brains will be removed after a quick decapitation and dipped in the PBS slush (0.1 M, pH 7.4) to remove surface blood. Once the brain tissue is frozen in dry ice, it can be wrapped in labeled foil and stored at -80 °C.  
  2. Sixteen micron thickness frozen sections will be cut in a cryostat, collected on slides and stored at -20 °C freezer for less than 48 h.
  3. Slides will be brought to room temperature on the experiment day; DO NOT open the box until slides have equilibrated to room temperature (about 20 min at room temperature) in order to avoid excessive moisture on tissue sections. Afterwards, slides will be selected to be used for the experiment.
  4. Wash with 50 mM Tris-Citrate buffer (pH 7.1) at 4 °C for 5 min three times.
  5. Let slides dry at room temperature (leave slides for 45 min to 1 h or longer).
  6. While waiting for the slides to dry
    1. Make “Shoot for” solutions:
      1. Theoretical “Shoot for”: “Shoot for” is the stock solution used to make a series of dilutions for receptor saturation analysis. The range of [3H]gaboxadol concentrations will be determined based on the literature and previous experiments. The highest concentration will then be used to calculate a theoretical “Shoot for”. For example, if the highest concentration used in the experiment is 400 nM of [3H]gaboxadol, the total volume added to the slide mailer is 7 ml.  The volume of the 10X stock displacer solution to be added to the mailer is 0.7 ml; therefore, 6.3 ml of X concentration of the “Shoot for” will be needed. Thus, the “Shoot for” will be calculated as:
        X = (400 nM*7 ml)/6.3 ml, and X = 444.44 nM.
      2. Volume (V) of the original  [3H]gaboxadol required to make the theoretical “Shoot for”:     
        V (μl) = Vt * X * SA
        SA is the radioactive ligand’s specific activity (Ci/mmol) provided by the manufacturer. In addition, the specific activity for the same ligand may vary between production batches;
        X is the theoretical concentration of the “Shoot for” (nM);
        Vt is the total volume of “Shoot for” needed to make the series dilution (L). Therefore, Vt = V1+V2+……+VHighest
        For each concentration, there will be a “total” mailer (measuring total binding, displacer omitted) and a “blank” mailer (measuring non-specific binding, displacer added). For example, a series of concentrations of 5, 10, 25, 50, 75, 100, 250 and 400 nM [3H]gaboxadol are used and the total volume of the “Shoot for”:
         Vt (L) = [(5 nM*7 ml/X)+(10 nM*7 ml/X)… +(400 nM*7 ml/X)]*2    
                        1000  
      3. The “Shoot for” solution will be made by adding V (μl) of the original [3H]gaboxadol to Vt-V amount of the incubation buffer.
    2. Count “Shoot for” using Scintillation counter by aliquoting 10 μl of “shoot for” into 5 ml of scintillation fluid. Based on the counts, calculate the actual concentration of the “Shoot for” using GraphPad Radioactive Calculator. If the measured value is close to the theoretical concentration, continue to the next step. From this point forward, the actual concentration of the stock solution will be used for the dilution calculations.
    3. Pipetting required amount of 50 mM Tris-Citrate buffer to tubes labeled with Total or Blank of 5, 10, 25, 50, 75, 100, 250 and 400 nM of [3H]gaboxadol, add appropriate volume of the stock solution to make to the designated concentrations.
    4. Add 0.7 ml of 200 μM GABA into tubes labeled Blank.
    5. Count the counts of the mixture in 16 tubes. The actual concentration of [3H]gaboxadol in each tube will be calculated based on these counts.
  7. Transfer mixtures from 16 tubes into the correctly labeled slide mailer.
  8. Drop slides into their designated mailers. Shake well, before leaving mailers in the refrigerator for 1 h at 4 °C.
  9. After incubation, slides will be washed twice in cold Tris-Citrate buffer for 10+10 sec, followed by cold dH2O for 10 sec.
  10. Let slides dry for 2-4 h or overnight.
  11. Break the slides: re-label the slides next to the sections, line up sections with a ruler and cut the slides using a diamond scribe. The slide end (with one section on it) will be taped using double-sided tape after being broken.
  12. Put the slides onto the phosphor transcreens; transcreen exposure time is about 48 h.
  13. Binding images are obtained by scanning the exposed phosphor transcreens using a Cyclone Phosphor Imager.


    Figure 1. An autoradiographic image of [3H]gaboxadol  on GABAA receptor binding in a four month old Fischer Brown Norway rat brain tissue (16 μm thickness). Highest binding area (circled) is the medial geniculate body, while hippocampus and cortex show moderate binding.

Recipes

  1. Binding mixture
    Binding mixture consists of 50 mM Tris-Citrate buffer and [3H]gaboxadol with/without 200 μM GABA

Acknowledgments

This protocol is adapted from Richardson et al. (2011) and Richardson et al. (2013). Work supported by NIH DC000151 to DMC.

References

  1. Richardson, B. D., Ling, L. L., Uteshev, V. V. and Caspary, D. M. (2013). Reduced GABA(A) receptor-mediated tonic inhibition in aged rat auditory thalamus. J Neurosci 33(3): 1218-1227a.
  2. Richardson, B. D., Ling, L. L., Uteshev, V. V. and Caspary, D. M. (2011). Extrasynaptic GABA(A) receptors and tonic inhibition in rat auditory thalamus. PLoS One 6(1): e16508.
  3. Storustovu, S. I. and Ebert, B. (2006). Pharmacological characterization of agonists at delta-containing GABAA receptors: Functional selectivity for extrasynaptic receptors is dependent on the absence of gamma2. J Pharmacol Exp Ther 316(3): 1351-1359.

材料和试剂


  1. [来自Merck& Co.的礼品]
  2. GABA(Sigma-Aldrich,目录号:A2129)
  3. 孵育缓冲液:50mM Tris-柠檬酸盐缓冲液(pH7.1)
  4. 0.1M PBS(磷酸盐缓冲盐水,pH7.4)结合混合物(参见配方)

设备

  1. 幻灯片(Fisher Scientific,型号:HS15986)
  2. 标签箔
  3. 低温恒温器(Leica Microsystems,型号:CM1850)
  4. -20℃冰箱
  5. 闪烁计数器
  6. 标尺
  7. 钻石抄写员
  8. 双面胶带
  9. 磷光转换器
  10. 旋风磷光成像仪(PerkinElmer)

软件

  1. OptiQuant 4.0(PerkinElmer)
  2. GraphPad放射性计算器(GraphPad软件)

程序

  1. 在快速断头后取出动物大脑,并浸入PBS浆液(0.1M,pH7.4)中以除去表面血液。 一旦脑组织在干冰中冷冻,可将其包裹在标记的箔中并储存在-80℃下 。  
  2. 在低温恒温器中切割16微米厚度的冷冻切片,在载玻片上收集,并在-20℃冷冻箱中保存小于48小时。
  3. 在实验当天,玻片将达到室温; 不要打开包装盒,直到载玻片平衡到室温(在室温下约20分钟),以避免组织切片上过多的水分。 之后,将选择幻灯片用于实验。
  4. 用50mM Tris-柠檬酸盐缓冲液(pH7.1)在4℃洗涤5分钟3次
  5. 让载玻片在室温下干燥(将玻片保持45分钟至1小时或更长时间)
  6. 在等待幻灯片干燥的同时
    1. 制作"拍摄"解决方案:
      1. 理论"拍摄":"拍摄"是用于进行一系列稀释用于受体饱和分析的储备溶液。 [sup] 3 H]加波沙朵浓度的范围 基于文献和先前的实验确定。然后将使用最高浓度来计算理论"拍摄"。例如,如果实验中使用的最高浓度为400nM的[3 H]加波沙朵,则添加到载玻片邮包中的总体积为7ml。加入到邮包中的10X原液置换剂溶液的体积为0.7ml;因此,需要6.3ml的 X 浓度的"Shoot for"。因此,"Shoot for"将计算为:
        X =(400 nM * 7 ml)/6.3 ml, X = 444.44 nM。
      2. 原件的体积(V) [ 3 H]加波沙朵使理论"拍摄":   
        V (μl)= V t * X < SA是由制造商提供的放射性配体的比活性(Ci/mmol)。此外,相同配体的比活性在生产批次之间可能不同;
        X 是"Shoot for"的理论浓度(nM);
        V t 是进行系列稀释所需的"Shoot for"的总体积(L)。因此, V t = V 1 + V 2 最高
        对于每个浓度,将存在"总"邮递(测量总结合,置换剂省略)和"空白"邮递(测量非特异性结合,置换剂添加)。例如,使用5,10,25,50,75,100,250和400nM的一系列浓度的[加3 H]加波沙朵,并且"Shoot for"的总体积: br /> </strong>(L)= [(5nM * 7ml/X)+(10nM * 7ml/X) X)... +(400nM * 7ml/X)] * 2</u>  
                         1000 
      3. 通过向 V t 添加 V (μl)原始[3H]加波沙朵,/sub> - V 孵育缓冲液
    2. 计数"拍摄"使用闪烁计数器通过等分10微升"拍摄"到5毫升闪烁液。基于计数,使用GraphPad Radioactive Calculator计算"Shoot for"的实际浓度。如果测量值接近理论浓度,则继续下一步。从这一点开始,原液的实际浓度将用于稀释计算
    3. 将所需量的50mM Tris-柠檬酸盐缓冲液吸移到用总量或空白5,10,25,50,75,100,250和400nM的[3 H]加波沙朵标记的试管中,加入适当的使储液的体积达到指定浓度
    4. 向标记为空白的管中加入0.7ml200μMGABA。
    5. 计数混合物在16管中的计数。将基于这些计数计算每个试管中的[
  7. 将混合物从16个管中转移到正确标记的幻灯片邮件中。
  8. 将幻灯片放入指定的邮件。 摇匀,然后将邮件在冰箱中在4℃下放置1小时
  9. 孵育后,将载玻片在冷Tris-柠檬酸盐缓冲液中洗涤两次,持续10 + 10秒,然后冷却dH 2 O 10秒。
  10. 让幻灯片干燥2-4小时或过夜。
  11. 打开幻灯片:将部分旁边的幻灯片重新标记,用标尺对齐部分,然后使用金刚石划片切割幻灯片。 滑动端(带有一个部分)在断裂后将使用双面胶带进行录制。
  12. 将幻灯片放在荧光屏上; 转盘曝光时间约为48小时
  13. 通过使用Cyclone Phosphor Imager扫描曝光的荧光体转换屏获得结合图像

    图1. [ 3 H]加波沙朵的放射自显影图像 在四个月龄的Fischer Brown Norway大鼠脑组织(16μm厚度)中的GABA A受体结合。最高结合面积(圆圈)是内侧膝状体,而海马和皮质显示中度结合。

食谱

  1. 结合料
    结合混合物由50mM Tris-柠檬酸盐缓冲液和含/不含200μMGABA的[3 H]加波沙朵组成

致谢

该协议改编自Richardson等人(2011)和Richardson等人(2013)。 NIH DC000151到DMC支持的工作。

参考文献

  1. Richardson,B.D.,Ling,L.L.,Uteshev,V.V.and Caspary,D.M。(2013)。 减少老年大鼠听觉丘脑中GABA(A)受体介导的强直抑制。 em> J Neurosci 33(3):1218-1227a。
  2. Richardson,B.D.,Ling,L.L.,Uteshev,V.V.and Caspary,D.M。(2011)。 突触外GABA(A)受体和大鼠听觉丘脑的强直抑制。 PLoS One 6(1):e16508。
  3. Storustovu,S.I。和Ebert,B。(2006)。 含δGABA A受体激动剂的药理学表征:突触后受体的功能选择性取决于缺失 的γ2。 Pharmacol Exp Ther 316(3):1351-1359。
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How to cite this protocol: Ling, L. and Caspary, D. (2013). Autoradiographic 3H-Gaboxadol Receptor Binding Protocol. Bio-protocol 3(23): e989. DOI: 10.21769/BioProtoc.989; Full Text



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