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The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the “hippocampus proper” which consists only of CA1, CA3 and smaller CA2 and CA4 areas, from the “hippocampal formation,” composed of these in addition to the dentate gyrus and subiculum). Distinct cell types give rise to unique axonal fiber pathways in the dentate gyrus, CA3 and CA1 subregions; accordingly, these areas may exhibit differential molecular profiles in response to a number of behavioral paradigms and pharmacological and genetic treatments. It is therefore in the interest of the investigator to dissect a specific subregion from the whole hippocampus. Here we outline a protocol for subregion-specific dissection from the adult mouse.

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Dissection of Different Areas from Mouse Hippocampus
小鼠海马体不同部位的解剖

神经科学 > 行为神经科学 > 认知
作者: Faraz A. Sultan
Faraz A. SultanAffiliation: Neurobiology Department, University of Alabama at Birmingham, Birmingham, USA
For correspondence: faraz@uab.edu
Bio-protocol author page: a953
Vol 3, Iss 21, 11/5/2013, 6781 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.955

[Abstract] The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the “hippocampus proper” which consists only of CA1, CA3 and smaller CA2 and CA4 areas, from the “hippocampal formation,” composed of these in addition to the dentate gyrus and subiculum). Distinct cell types give rise to unique axonal fiber pathways in the dentate gyrus, CA3 and CA1 subregions; accordingly, these areas may exhibit differential molecular profiles in response to a number of behavioral paradigms and pharmacological and genetic treatments. It is therefore in the interest of the investigator to dissect a specific subregion from the whole hippocampus. Here we outline a protocol for subregion-specific dissection from the adult mouse.

Keywords: Hippocampus(海马), Memory(记忆), Learning(学习), CA1(CA1)

[Abstract]

Materials and Reagents

  1. Microspatula (Thermo Fisher Scientific, catalog number: 21-401-10 )
  2. Short microspatula (Thermo Fisher Scientific, catalog number: 21-401-15 )
  3. Filter paper (11 cm) (Thermo Fisher Scientific, catalog number: 09-795D )
  4. WypAll or KimWipes
  5. Ice buckets
  6. Cloth diapers
  7. Dry Ice
  8. Biohazard waste bags
  9. Adult laboratory mice
  10. 10x Stock Cutting Solution (see Recipes)
  11. 1 M KCl (see Recipes)
  12. 1 M MgCl2 (see Recipes)
  13. 1 M CaCl2 (see Recipes)
  14. 1x Cutting Solution (make from 10x stock) (see Recipes)

Equipment

  1. Surgical Scissors (Fine Science Tools, catalog number: 14130-17 )
  2. Fine Scissors (Fine Science Tools, catalog number: 14094-11 )
  3. Forceps (Fine Science Tools, catalog number: 11506-12 )
  4. Scalpel handle (Fine Science Tools, catalog number: 10003-12 )
  5. Scalpel blades #15 (Fine Science Tools, catalog number: 10015-00 )
  6. Single edge razor blades
  7. Petri dishes (100 mm x 15 mm) (Sigma-Aldrich, catalog number: CLS3160101 )
  8. Transfer pipettes (Thermo Fisher Scientific, catalog number: 13-711-7M )
  9. 20 ml beaker (Thermo Fisher Scientific, catalog number: 02-539-1 )
  10. 600 ml beaker (Sigma-Aldrich, catalog number: CLS1000600 )
  11. 1 L bottle (Sigma-Aldrich, catalog number: CLS13951L )
  12. Nuclease-free microtubes (1.5-1.7 ml)
  13. Plastic spoon
  14. Microscope
  15. Pressurized oxygen tank (95% O2/5% CO2) with tubing

Procedure

  1. Place container of 1x Cutting Solution in a filled ice bucket, and transfer ~15 ml of the solution into a 20 ml beaker also on wet ice.
  2. Oxygenate the solutions in both the bottle and beaker for at least 20 min before beginning (see below).
  3. Surround bench with cloth diapers, and secure a biohazard waste bag near the work bench.




  4. Place all dissection tools except razor blades and scalpel into a 600 ml beaker containing sterile water and WypAll or KimWipe cloths submerged in the water. Place the tools facing down on the cloths so as to minimize direct contact with the glass beaker.
  5. Place labeled microtubes in a bucket containing dry ice (blue bucket above). Likewise, place one piece of a petri dish on the dry ice and let cool.
  6. Place small ice bucket containing ice under microscope (purple bucket above).
  7. Fill one ice bucket with ice and secure the other piece of the petri dish on the ice so that the edges are facing down. Place one piece of filter paper on the dish.
  8. Immediately before beginning, transfer enough cutting solution from the 20 ml beaker to the filter paper and spread evenly.
  9. Place a new cloth diaper over the bench before each dissection. Place the mouse on the diaper, maintaining a grip over the tail with the right hand (for right-handed individuals).
  10. Rapidly secure the mouse with the left hand such that the scruff posterior to the rodent’s head is firmly held between the left thumb and proximal forefinger. The mouse should be firmly in the experimenter’s control at this point. Transfer the base of the tail from the right hand to the left hand so that it is secured between the distal ring and little fingers.
  11. Place smooth end of large scissors just behind the skull and hold down firmly with the right hand. Rapidly pull on the tail with the left hand until the rodent skull is dislocated from the spinal cord.
  12. After cervical dislocation, decapitate with the same scissors, being sure to cut just behind the skull. If the cut is too far posterior, excess tissue will occlude the foramen magnum.
  13. While gently pulling the scalp to lateral sides, cut scalp skin from between the rodent’s eyes down the midline using a razor blade.



  14. Place one tip of the fine scissors into the foramen magnum and cut laterally into the skull. Repeat for the other side. Gently cut from the same cavity up the midline towards the nose, trying to keep the end of the scissors as superficial as possible so as not to perturb the brain.



  15. Make small cuts from the midline incision near the eyes laterally.
  16. Use forceps to apply gentle tangential, lateral pressure to either of the newly formed skull flaps. Repeat for the remaining side. If force is properly applied, the skull should be fully removed and the brain exposed. If a piece of skull breaks off before the respective hemisphere is exposed, discard it and use forceps again to coerce the remaining flap to the side. In this case, be sure to minimize contact between the forceps tips and brain tissue.



  17. Use longer microspatula to gently transfer the brain to the 20 ml beaker of oxygenated cutting solution. This involves tearing cranial nerve fibers near the base of the brain.



  18. Use the spoon to transfer the brain to the wetted filter paper, and hemisect the brain using a clean razor blade. Transfer either hemisphere back to the 20 ml beaker.
  19. Using the two short microspatulas, remove the hippocampus: Anchor one spatula tip just over the cerebellum near the junction with the cortex. Place the other spatula tip near the same junction and peel the cortical hemisphere laterally in a gentle manner. Periodically use the transfer pipette to apply fresh cutting solution to the tissue.



  20. Once the cortical hemisphere is fully peeled laterally, the hippocampus should be exposed. While anchoring the brain with one spatula tip, place the other just under the caudal tip of the hippocampus. Carefully apply pressure to the medial white matter tracts with the anchoring spatula while moving the second spatula tip slightly anteriorly and laterally. “Scoop” or “roll” the hippocampus laterally with that spatula tip. If done properly, the hippocampus should land on the filter paper with “glossy” end up. Repeat for the second hemisphere.
  21. Once both hippocampi are removed and placed on the filter paper, transfer it along with the petri dish piece to the ice bucket underneath the microscope. Align each hippocampus so that the anterior end (slightly thinner than the posterior end – this is more easily seen by observing the hippocampus from the side rather than the top) is facing upward.



  22. Use the scalpel to make small vertical incisions in both the anterior and posterior tips of the hippocampus. This will create flat, blunt tips.
  23. Make a similar incision about 1/3rd or 1/4th of the way from the anterior to posterior end while observing through the microscope. Carefully “stand” the small segment of the tissue above the blade on the face created earlier (right figure below – CA1 is on top). Be sure to apply fresh cutting solution but only in small quantities – excess solution will blur the distinctions of the hippocampal subregions.



  24. Using the scalpel, dissect out area CA1, CA3 or dentate gyrus (DG) with one curved cut downwards through the tissue (see diagram below for approximate dissection guidelines for a right hippocampus cross-section). Immediately transfer the dissected tissue with the scalpel to the petri dish piece on dry ice. The tissue should freeze very rapidly.



  25. Repeat step 23 with the remaining piece of tissue, keeping in mind that CA1 ends approximately 2/3rds of the way from the anterior to posterior end. Depending on the thickness of pieces cut, a total of 2-4 segments will have been created. Work quickly and minimize overall disruption of brain tissue.
  26. Once all pieces of tissue are frozen on dry ice, use the scalpel blade to detach the tissue from the petri dish and place in a labeled microtube. It is often convenient to “stack” tissue on the petri dish as it is collected. This way, one larger piece will be created, and it can be dislodged more easily than multiple small pieces of frozen tissue.
  27. Store microtubes at -80 °C until tissue is ready for processing.

Recipes

  1. 10x Stock Cutting Solution
    For 1 L stock, mix the following in a graduated cylinder and bring up to 1 L with sterile Milli-Q water:
    73.00 g NaCl
    1.72 g Sodium phosphate (monobasic, monohydrate)
    21.00 g Sodium bicarbonate
    18.10 g D-Glucose
    Sterile filter with vacuum filtration, and stored at 4 °C
  2. 1 M KCl
    Dissolve 14.91 g KCl in Milli-Q water and bring up to a volume of 200 ml
    Sterile filter and stored at 4 °C
  3. 1 M MgCl2
    Dissolve 40.66 g MgCl2 hexahydrate in Milli-Q water and bring up to a volume of 200 ml
    Sterile filter and stored at 4 °C
  4. 1 M CaCl2
    Dissolve 29.40 g CaCl2 dihydrate in Milli-Q water and bring up to a volume of 200 ml
    Sterile filter and stored at 4 °C
  5. 1x Cutting Solution
    For 1 L solution:
    Mix 100 ml 10x stock solution
    120 mg sodium L-ascorbate
    3 ml 1 M KCl stock, bring up to 900 ml with Milli-Q water
    Begin oxygenation with 95% O2/5% CO2 for at least 20 minutes
    Add 7 ml MgCl2 and 0.5 ml CaCl2 and continue oxygenation
    Confirm that pH is in physiological range ~7.2-7.4
    Bring up to a final volume of 1 L with Milli-Q water
    (Optional) Confirm that osmolarity is ~315-325 mOsm
    Sterile filter, keep container on ice and bring to experiment setup

Acknowledgments

This protocol was adapted from a previously published paper: Levenson et al. (2006). This work was supported by the NIH (MH095270, MH57014, AG031722, NS057098, P30 NS47466), the Ellison Medical Foundation, and the McKnight Brain Research Foundation.

References

  1. Levenson, J. M., Roth, T. L., Lubin, F. D., Miller, C. A., Huang, I. C., Desai, P., Malone, L. M. and Sweatt, J. D. (2006). Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 281(23): 15763-15773.
  2. Miller, C. A. and Sweatt, J. D. (2007). Covalent modification of DNA regulates memory formation. Neuron 53(6): 857-869.
  3. Sultan, F. A., Wang, J., Tront, J., Liebermann, D. A. and Sweatt, J. D. (2012). Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity. J Neurosci 32(48): 17059-17066.        
  4. Sweatt, J. D. (2009). Mechanisms of Memory. Academic Press.

材料和试剂

  1. Microspatula(Thermo Fisher Scientific,目录号:21-401-10)
  2. 短小芽(Thermo Fisher Scientific,目录号:21-401-15)
  3. 滤纸(11cm)(Thermo Fisher Scientific,目录号:09-795D)
  4. WypAll或KimWipes
  5. 冰桶
  6. 布尿布
  7. 干冰
  8. 生物危险废物袋
  9. 成人实验室小鼠
  10. 10x切割解决方案(参见配方)
  11. 1 M KCl(见配方)
  12. 1 M MgCl <2> (参见配方)
  13. 1 M CaCl <2> (参见配方)
  14. 1x切割解决方案(从10x股票制造)(见配方)

设备

  1. 外科剪刀(Fine Science Tools,目录号:14130-17)
  2. Fine Scissors(Fine Science Tools,目录号:14094-11)
  3. 镊子(Fine Science Tools,目录号:11506-12)
  4. 手术刀柄(Fine Science Tools,目录号:10003-12)
  5. 手术刀片#15(Fine Science Tools,目录号:10015-00)
  6. 单刃剃刀刀片
  7. 培养皿(100mm×15mm)(Sigma-Aldrich,目录号:CLS3160101)
  8. 转移移液管(Thermo Fisher Scientific,目录号:13-711-7M)
  9. 20ml Thermo烧杯(Thermo Fisher Scientific,目录号:02-539-1)
  10. 600ml烧杯(Sigma-Aldrich,目录号:CLS1000600)
  11. 1L瓶(Sigma-Aldrich,目录号:CLS13951L)
  12. 无核酸酶的微管(1.5-1.7ml)
  13. 塑料勺
  14. 显微镜
  15. 用管子
    加压的氧气罐(95%O 2/5%CO 2)

程序

  1. 地点   容器的1x切割溶液在填充的冰桶,并转移 〜15ml溶液放入20ml烧杯中,也在湿冰上
  2. 在开始之前,将瓶子和烧杯中的溶液氧化至少20分钟(见下文)
  3. 用布尿布环绕工作台,并在工作台附近安装生物危害废物袋



  4. 地点   所有解剖工具除了剃刀刀片和解剖刀进入600毫升 烧杯含无菌水和WypAll或KimWipe布浸没 在水里。 将工具面朝下放在布上 最小化与玻璃烧杯的直接接触
  5. 地点 标记的微管在含有干冰(上面的蓝色桶)的桶中。 同样,将一块培养皿放在干冰上,让其冷却
  6. 在显微镜下放置装有冰的小冰桶(上面的紫色桶)
  7. 填   一个冰桶与冰和固定培养皿的另一片   冰使得边缘面向下。 放一块过滤器 纸上的菜。
  8. 在开始之前,将足够的切割溶液从20ml烧杯中转移到滤纸上并均匀铺展
  9. 地点   在每次解剖之前在板凳上的新的布尿布。 放置 鼠标在尿布上,保持在右边的尾巴上的抓地力 手(对于右撇子)
  10. 快速安全 鼠标用左手,使得耙齿在啮齿动物的后面 头部牢固地保持在左拇指和近端食指之间。 的 小鼠应该坚定地在实验者的控制在这一点。 将尾巴的底部从右手移动到左手 它被固定在远端环和小指之间
  11. 地点   光滑的大剪刀的末端在头骨后面坚定地坚持   用右手。 用左手快速拉尾,直到 啮齿动物的头骨从脊髓脱位
  12. 后 子宫颈脱位,用同样的剪刀断头,一定要 切在头骨后面。 如果切口太后,过多 组织将阻塞孔的大小。
  13. 轻轻地 将头皮拉到侧面,从两者之间切割头皮 啮齿动物的眼睛沿着中线使用剃刀刀片


  14. 地点   一把精细的剪刀插入孔中,并横向切开 进入头骨。 对另一侧重复。 轻轻地从同一个 空洞朝着鼻子的中线,试图保持结束 剪刀尽可能表面,以免干扰大脑


  15. 从侧面靠近眼睛的中线切口做小切口。
  16. 使用  钳子施加温和切向,侧向压力到任一 新形成的颅骨襟翼。对剩余的一侧重复。如果力是 适当应用,头骨应该完全清除和大脑 裸露。如果一块头骨在相应的半球之前断裂  暴露,丢弃它,并再次使用镊子强制剩余 翻边。在这种情况下,请确保最小化之间的接触 钳子尖和脑组织


  17. 使用更长的小芽轻轻地将大脑转移到20毫升烧杯  的氧合切割溶液。这涉及到撕裂颅神经 纤维靠近大脑底部。



  18. 使用勺子将大脑转移到润湿的滤纸上,和 使用干净的剃刀刀片半截脑。 转移任一半球   回到20毫升烧杯
  19. 使用两个短 去除海马:将一个刮刀尖固定在正上方 小脑在与皮层的连接点附近。 放置另一个 刮刀尖附近相同的连接处并剥离皮层半球 侧向以轻微的方式。 定期使用移液器 将新鲜切割溶液应用于组织


  20. 一旦皮质半球侧面完全剥离,海马 应该暴露。当用一个铲刀尖锚定大脑时,放置  另一个恰好在海马的尾端。小心申请  压力到内侧白质束与锚固刮刀 同时稍微向前和侧向移动第二铲刀尖端。 用该刮铲尖端横向地"舀"或"滚动"海马。如果 正确地做,海马应该着陆在滤纸上 "有光泽"结束。对第二个半球重复此步骤。
  21. 一旦两者  取出海马并置于滤纸上,转移它 随着培养皿片到下面的冰桶 显微镜。对齐每个海马,使前端(略 比后端更薄 - 这通过观察更容易看到 海马从侧面而不是顶部)朝上。



  22. 使用解剖刀在前面做小的垂直切口 和海马的后端。 这将创建平,钝 提示。
  23. 做一个类似切口约1/3 rd 或1/4 th 方式从前到后,同时通过观察 显微镜。 小心"站立"上面的组织的小段 刀片在前面创建的面上(右图--CA1在顶部)。 一定要使用新鲜的切割溶液,但只有少量 - 过量的溶液将模糊海马的区别 子区域


  24. 使用手术刀,解剖区域CA1,CA3或齿状回(DG) 一个弯曲向下穿过组织(见下图 近似解剖指南为右海马 横截面)。 立即将切开的组织转移 解剖刀到干冰上的培养皿片。 组织应该冻结 非常快。



  25. 重复   步骤23与剩余的组织片段,记住CA1 从前端到后端大约2/3 rds 。   根据切割的厚度,总共2-4段 已创建。 快速工作,尽量减少大脑的整体中断   组织
  26. 一旦所有的组织片都在干冰上冷冻, 使用手术刀刀片从培养皿和地方分离组织   在标记的微管中。 通常方便的是"堆叠"组织   培养皿,因为它是收集。 这样,一个更大的块将 创建,并且它可以比多个小块更容易被移除 的冷冻组织
  27. 将微管存放在-80℃下,直到组织准备好处理

食谱

  1. 10x库存切割解决方案
    对于1 L原液,将其混合在量筒中,用无菌Milli-Q水补足至1 L:
    73.00克NaCl
    1.72g磷酸钠(一碱价,一水合物)
    21.00克碳酸氢钠
    18.10g D-葡萄糖
    无菌过滤器用真空过滤,并在4℃下保存
  2. 1 M KCl
    将14.91g KCl溶解在Milli-Q水中并使体积达到200ml
    无菌过滤并在4℃下保存
  3. 1 M MgCl 2
    在Milli-Q水中溶解40.66g MgCl 2 6水合物并使体积达到200ml
    无菌过滤并在4℃下保存
  4. 1 M CaCl 2
    在Milli-Q水中溶解29.40g CaCl 2 2水合物,并使体积达到200ml
    无菌过滤并在4℃下保存
  5. 1x切割解决方案
    对于1 L溶液:
    混合100 ml 10x储备液
    120mg L-抗坏血酸钠 3 ml 1 M KCl原液,用Milli-Q水
    升至900 ml 用95%O 2/5%CO 2开始氧合至少20分钟
    加入7ml MgCl 2和0.5ml CaCl 2并继续充氧
    确认pH在生理范围〜7.2-7.4
    用Milli-Q水冲洗至最终体积为1 L,
    (可选)确认渗透压为〜315-325 mOsm
    无菌过滤器,保持容器在冰上,带来实验设置

致谢

该协议改编自先前发表的论文:Levenson等人(2006)。这项工作由NIH(MH095270,MH57014,AG031722,NS057098,P30 NS47466),Ellison医学基金会和McKnight脑研究基金会支持。

参考文献

  1. Levenson,J.M.,Roth,T.L.,Lubin,F.D.,Miller,C.A.,Huang,I.C.,Desai,P.,Malone,L.M。和Sweatt,J.D。(2006)。 DNA(胞嘧啶-5)甲基转移酶调节海马突触可塑性的证据。 em> J Biol Chem 281(23):15763-15773。
  2. Miller,C.A。和Sweatt,J.D。(2007)。 DNA的共价修饰调节记忆形成。 神经元 53 (6):857-869。
  3. Sultan,F.A.,Wang,J.,Tront,J.,Liebermann,D.A。和Sweatt,J.D。(2012)。 基因缺失的Gadd45b ,是一种活性DNA去甲基化的调节剂,增强了长-term memory and synaptic plasticity。 J Neurosci 32(48):17059-17066。        
  4. Sweatt,J.D。(2009)。 内存机制。 学术出版社
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sultan, F. A. (2013). Dissection of Different Areas from Mouse Hippocampus. Bio-protocol 3(21): e955. DOI: 10.21769/BioProtoc.955; Full Text
  2. Sultan, F. A., Wang, J., Tront, J., Liebermann, D. A. and Sweatt, J. D. (2012). Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity. J Neurosci 32(48): 17059-17066.        




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    (提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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