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Isolation and Maintenance of Striatal Neurons
小鼠胚胎纹状体神经元的分离与维持   

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Tatiana Rosado Rosenstock Tatiana Rosado Rosenstock
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本实验方案简略版
Molecular Neurobiology
Sep 2017

Abstract

Primary cultures of murine striatal neurons are widely used to explore cellular mechanisms in neurobiology, including brain diseases. Here we describe a detailed and standardized protocol to dissect and culture embryonic murine striatal neurons GABA-positive/DARPP-32-positive for 12 days in vitro, when they show good neuronal cell connectivity and the presence of dendritic spines, which reflects the maturation of the network.

Keywords: Primary cultures (原代培养物), Striatum dissection (纹状体解剖), Striatal neurons (纹状体神经元), Medium spiny neurons (中型多棘神经元)

Background

Striatum is a critical component of the motor and reward systems and dysfunction of striatal neurons can lead to a variety of neuronal disorders, from obsessive-compulsive-like behaviors (Welch et al., 2007) to neurodegeneration, as observed in Huntington’s disease (Reiner et al., 1988). Therefore, a well-stablished striatal culture may be a great value as a model for studying these and other conditions. The major neuronal subtype in adult striatum is medium spiny projection neurons (MSNs), which constitutes approximately 95% of all striatal neurons and use the inhibitory transmitter gamma-aminobutyric acid (GABA). They are characterized by medium-sized cell bodies, complex dendritic arbors and a high density of dendritic spines that receive both glutamatergic and dopaminergic inputs. The remaining 5% of neurons are composed by GABAergic aspiny interneurons (Graveland and DiFiglia, 1985; Matamales et al., 2009). The following protocol describes the preparation of primary cultures of mouse embryonic striatal neurons with a low percentage of astrocytes, which proliferation is prevented by the mitosis inhibitor 5-fluoro-2’-deoxyuridine (5-FdU). After 12 days in vitro (DIV) the striatal neurons obtained by the described protocol are expected to be immunolabeled for microtubule-associated protein 2 (MAP2) or NeuN, GABA and a large percentage for dopamine- and cAMP-regulated phosphoprotein (DARPP-32).

Materials and Reagents

  1. Coverslips 18 mm diameter round, #1.5 (0.17 mm) thickness (Thermo Fisher Scientific, Menzel, catalog number: 11817742 )
  2. Sterile non-tissue culture treated Petri dishes of 35, 60 and 100 mm (VWR, catalog numbers: 734-1707 , 734-1708 , 734-1709 )
  3. 5, 10 ml serological sterile pipettes (Labbox, catalog numbers: MPIP-U05-200 , MPIP-U10-200 )
  4. Sterile 0.2-10 μl, 10-200 μl and 100-1,000 μl micropipette tips (Frilabo, catalog numbers: 170558 , 171017 , 171024 )
  5. 15 and 50 ml sterile conical centrifuge tubes (Corning, catalog numbers: 430791 , 430829 )
  6. Sterile 1.5 ml microcentrifuge tubes (BIOplastics, catalog number: B74085 )
  7. Sterile syringes, 20 ml (Terumo, catalog number: SS+20L1 )
  8. Sterile 230 mm glass Pasteur pipettes (Labbox, catalog number: PIPN-230-250 )
  9. Sterile 0.2 μm acetate cellulose filters (GE Healthcare, Whatman, catalog number: 28415732 )
  10. Vacuum 0.2 μm filter bottle system, 500 ml (Corning, catalog number: 430769 )
  11. Cell strainers, 40 μm (Corning, Falcon®, catalog number: 352340 )
  12. Multi-wells tissue culture treated, flat bottom, polystyrene (Corning, catalog numbers: 3596 , 3548 , 3524 , 3512 , 3506 )
  13. Pregnant female mice (Mus musculus) with 16 days of gestation
    Note: In this protocol, mice from FVB/NJ inbred strain (THE JACKSON LABORATORY) were used.
  14. Hydrochloric acid (HCl), 37% (Sigma-Aldrich, catalog number: 435570 )
  15. Ethanol absolute 99.8% (Fisher Scientific, catalog number: 10342652 )
  16. Isoflurane Iso-Vet 1000 mg/g Inhalation Vapour (Chanelle UK)
  17. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  18. Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
  19. 5-FdU (Sigma-Aldrich, catalog number: F0503 )
  20. Anti-MAP2 (1:500; Santa Cruz Biotechnology, catalog number: sc-32791 )
  21. Anti-GABA (1:500; Sigma-Aldrich, catalog number: A2052 )
  22. Anti-DARPP32 (1:100; Abcam, catalog number: ab40801 )
  23. Goat anti-rabbit IgG secondary antibody, Alexa Fluor 594 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37117 )
  24. Donkey anti-mouse IgG secondary antibody, Alexa Fluor 488 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37114 )
  25. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  26. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P1149 )
  27. Potassium chloride (KCl) (Labbox, catalog number: POCL-00P-1K0 )
  28. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  29. Sodium chloride (NaCl) (Merck, catalog number: 106404 )
  30. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  31. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
  32. D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
  33. Sodium pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  34. HEPES (Sigma-Aldrich, catalog number: H3375 )
  35. Phenol red (Sigma-Aldrich, catalog number: P3532 )
  36. Deoxyribonuclease I (DNase I) (Sigma-Aldrich, catalog number: D5025 )
  37. Bovine serum albumin (BSA) fatty acid free (Sigma-Aldrich, catalog number: A6003 )
  38. Trypsin, Type IV-S, from porcine pancreas (Sigma-Aldrich, catalog number: T0303 )
  39. Trypsin inhibitor, type II-S: Soybean (Sigma-Aldrich, catalog number: T9128 )
  40. L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  41. Gentamicin (50 mg/ml) (Thermo Fisher Scientific, catalog number: 15750060 )
  42. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  43. B-27 supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  44. Poly-D-lysine for coating (0.1 mg/ml) (see Recipes)
  45. Hanks’ balanced salt solution (HBSS) (see Recipes)
  46. DNase I solution (5 mg/ml) (see Recipes)
  47. BSA solution (3 mg/ml) (see Recipes)
  48. Trypsin solution (0.5 mg/ml) (see Recipes)
  49. Trypsin inhibitor solution (1 mg/ml) (see Recipes)
  50. Washing solution (see Recipes)
  51. Inactivated FBS (see Recipes)
  52. Supplemented Neurobasal medium (see Recipes)

Equipment

  1. 11.5 cm fine straight scissor (Fine Science Tools, Dumont, catalog number: 14060-11 )
  2. 8.5 cm mini-dissecting scissor, straight with sharp tip (World Precision Instruments, catalog number: 503669 )
  3. Medium forceps (Fine Science Tools, model: Dumont #7, catalog number: 11274-20 )
  4. Forceps with straight and angled fine tip (Fine Science Tools, models: Dumont #5 and Dumont #5/45 )
  5. Automatic pipettor and micropipettes
  6. Glass Schott laboratory bottle with cap, 500 ml (DWK Life Sciences, Duran, catalog number: 21 801 44 5 )
  7. Water bath with electronic temperature regulation
  8. Ultrasonic cleaning bath (just for coverslips washing)
  9. Zeiss Stemi magnification glass (or equivalent)
  10. Phase contrast inverted microscope equipped with 10x and 20x objectives
  11. Zeiss LSM 710 point-scanning confocal microscope (ZEISS, model: LSM 710 or equivalent)
  12. Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  13. Water jacketed CO2 incubator
  14. Vertical laminar flow chamber
  15. Isoflurane vaporizer apparatus (E-Z Anesthesia, model: EZ-SA800 , or equivalent)

Software

  1. Fiji software (ImageJ, National Institute of Health, USA)

Procedure

Note: Except for the coverslips washing (described in the following Procedure A, before poly-D-lysine coating) and brain dissection, all the procedures must be done in a sterile environment using a flow chamber.

  1. Coverslips washing (timing ~20 h; before poly-D-lysine coating)
    1. Heat coverslips in a loosely covered glass bottle embedded in 1 M HCl at 50-60 °C for 4-16 h with occasional swirling. Use few coverslips at a time to prevent them from sticking each other. Acid washing protocol is recommended by the manufacturers for both enhancing the cleanliness of the coverslip and increasing cellular attachment.
    2. Cool at room temperature and carefully decant the HCl solution into an appropriate container. Rinse thoroughly with ddH2O and fill the glass bottle with ddH2O. Sonicate in a water bath for 30 min. Repeat the sonication step twice.
    3. Replace the ddH2O for a solution of 50% ethanol (v/v) and sonicate in a water bath for 30 min. Repeat the sonication step twice with an increasing concentration of ethanol: (i) 70% ethanol (v/v), (ii) 95% ethanol (v/v). Coverslips can be stored submerged in ethanol absolute ≥ 99.8% (v/v) for up to 6 months.
    4. Before use, sterilize the coverslips exposing them to UV light for ~15 min or by dry-heat sterilization.

  2. Poly-D-lysine coating (timing ~2.5 h; 1 day before culture)
    1. Add the previously prepared 0.1 mg/ml poly-D-lysine solution to the plates/coverslips desired for the culture until it covers the bottom. Incubate the plates/coverslips for at least 2 h, preferably overnight, at 37 °C in a humidified incubator.
    2. Aspirate the poly-D-lysine with a Pasteur pipette and rinse twice with sterile water. Remove the water and allow the well to dry in the laminar flow chamber.
    3. Store at 4 °C for up to 1 month. 

  3. Brain and striatum dissection (timing 1 h 30 min)
    1. Soak all the dissecting tools in 70% ethanol (v/v) before starting the dissection. Also, during the dissection routinely dip the dissecting tools in 70% ethanol (v/v) to reduce contaminations. Dry thoroughly before using.
    2. Anesthetize the pregnant female mice at gestational day 16 with 5% isoflurane (v/v) and sacrifice it by cervical dislocation (or choose an appropriate anesthesia according to the recommendations of the Institutional Animal Care and Use Committee ruling in the respective country of research). Place the mouse on its belly up and thoroughly rinse abdomen with 70% ethanol (v/v). Collect the embryos by cesarean-section by grasping the uterus with sterile forceps and cutting the connection with the mesometrium. Put the embryos in an HBSS-filled 100 mm Petri dish.
    3. Remove each embryo from the embryonic sac and rapidly decapitate it. Transfer the embryos’ heads to an HBSS-filled 60 mm Petri dish and keep it on ice.
    4. Using a pair of forceps, hold the head of the embryo and insert the tip of a straight Dumont #5 forceps between eyes to keep the head fixed above the surface. With the mini dissecting scissor pointed up, begin to cut the skin and skull from the end of the spinal cord (which is visible after decapitation) through the longitudinal fissure until the end of the head cavity. Support the head in the Petri dish and gently squeeze the brain out from the brain cavity in a frontal-occipital direction using an angled Dumont #5/45 forceps. Transfer one brain at a time to a new HBSS-filled 60 mm Petri dish (see Video 1).

      Video 1. Exemplification of how to remove the mouse brain from the skull

    5. Turn on the magnifying glass and adjust it for approximately 25x magnification. With the brain faced upward, start the dissection by making a sagittal cut approximately 1 mm parallel to the midline using the angled Dumont #5/45 forceps. Use this cut to facilitate the removal of the meninges from the cortex (see Video 2/Figures 1A-1D). To stabilize the brain during dissection, use a straight Dumont #5 forceps to gently hold the brain between the forebrain and the cerebellum.

      Video 2. Isolation of striatum from embryonic E16 mouse brain

    6. Open the slit with the help of the same angled forceps to unfold the anterior cortex overlaying the lateral ventricle and the ganglionic eminences. The striatum, presenting an irrigated oval structure, is located in the lateral eminence. Use the tips of the angled forceps to cut out the striatum pinching around, as shown in Video 2/Figures 1E-1F. Transfer it to a sterile HBSS-filled 35 mm Petri dish.
    7. Repeat the procedure on the other lobe and for each brain until all striata are collected. The remaining tissue can be harnessed to isolate the hippocampus, the cortex or other brain areas of interest.


      Figure 1. Steps for striata dissection from embryonic E16 mouse brains. A and B. Dorsal positioned embryos brains (C) are cut parallel to the central plane of the sagittal suture. Hold the cerebellum (Ce) with a straight Dumont #5 forceps to keep the brain stable during dissection. D. Using the open slit pull out the meninges (asterisk, *), which cover the whole forebrain (Fb). Afterward, use an angled Dumont #5/45 forceps (E) to unfold the anterior cortex (Cx) and (F) remove the striatum (St), next to the lateral ventricle, with a typical striated appearance that is not always visible at 25x magnification. Ob, olfactory bulbs; Bs, brainstem.

  4. Dissociation and plating of striatal cells (timing 30 min)
    1. Transfer the striata into sterile HBSS medium (7 ml) to a 15 ml-Falcon.
    2. Add 3 ml of trypsin solution and mix by gently inverting the tube. Incubate for 2-3 min in a 37 °C water bath gently mixing from time to time. Note that depending on the amount of tissue and the trypsin activity the trypsinization time may change. The current protocol is optimized to 3 ml of trypsin solution for about 6 embryos; an increased number of embryos may require an adjustment of the total volume of the solution, maintaining the trypsin ratio.
    3. Inactivate the trypsin by adding 5 ml of trypsin inhibitor solution. Note that if the volume added in the previous step is changed, the volume of trypsin inhibitor solution should be added accordingly. Mix by gently inverting the tube and wait until tissue settle. Aspirate the medium.
    4. Wash the cells by adding 5 ml of washing solution (trypsin inhibitor in BSA solution, 1:5) and mix by inverting the tube. Allow tissue to settle and aspirate as much volume as possible without disturbing the pellet.
    5. Add 2-3 ml of supplemented Neurobasal medium with 0.5% FBS and gently homogenize with P1000 pipette until the solution is homogeneous. Note that if trypsinization step is adequate, an aggressive trituration is not required, as too much physical stress on cells during mechanical homogenization will reduce cell viability.
    6. Prepare a 1.5 ml Eppendorf with 20 μl of cell suspension, 20 μl of Neurobasal medium and 20 μl of trypan blue solution, creating a 3-fold dilution factor. Use an inverted phase contrast microscope to count the number of viable cells in a hemocytometer and plate at a density of 130 x 103 cells/cm2. For low density cultures in coverslips plate 92 x 103 cells/cm2. Neurons are maintained at 37 °C in a humidified incubator with 5% CO2/95% air, for 12 days.
    7. At DIV3 remove one-third of culture medium and add an equal part of volume of fresh supplemented Neurobasal medium without FBS and containing 15 μM 5-FdU (final concentration of 5 μM) to reduce dividing non-neuronal cells in primary culture (Hui et al., 2016). Under these conditions, we observed approximately 15% of Glial fibrillary acidic protein (GFAP)-positive cells (data not shown). At DIV7 change the medium again by replacing half of the culture medium to fresh supplemented Neurobasal medium devoid of FBS and 5-FdU.
    8. Characterize your primary striatal culture in terms of percentage of neurons (MAP2 or NeuN-positive), glial cells (GFAP or S100 beta-positive), GABAergic cells (GAD65 or GABA-positive), MSNs (DARPP-32-positive and GAD65 or GABA-positive) and GABAergic interneurons (GAD65-positive and DARPP-32-negative). Figure 2 shows a representative image of a primary striatal neuronal culture with approximately 82% of GABA-positive plus DARPP-32-positive striatal neurons. 


      Figure 2. Characterization of mouse primary striatal culture. DIV12 cultured striatal neurons were stained with anti-MAP2 (1:500; Santa Cruz Biotechnology) antibody conjugated with secondary Alexa Fluor 594 (1:200; Ex: 590 nm, Em: 617 nm) together with (A) anti-GABA (1:500; Sigma-Aldrich) or (B) anti-DARPP32 (1:100; Abcam) antibodies conjugated with secondary Alexa Fluor 488 (1:200; Ex: 495 nm, Em: 519 nm). A secondary antibody-only control staining was performed, which showed no signal. Z-stack confocal images obtained with a 20x objective, NA=1.4 on an inverted confocal microscope. Scale bars = 50 μm.

Data analysis

For striatal cultures characterization, 6 to 8 images per individual experiment were randomly taken with confocal microscope for each antibody labeled. GABA- and DARPP-32-positive cells were counted using Fiji software (ImageJ, National Institute of Health, USA) with Cell Counter plugin, and the percentage were calculated in relative to MAP2-positive cells.

Recipes

  1. Poly-D-lysine for coating (0.1 mg/ml)
    1. Prepare a solution of 166 mM boric acid in type I (ultrapure, > 17.4 MΩ resistance) water and adjust the pH to 8.2
    2. Just before use, dilute the poly-D-lysine in the previous prepared boric acid solution and filter the solution in a laminar flow with a filtration unit
  2. Hanks’ balanced salt solution (HBSS)
    5.36 mM KCl
    0.44 mM KH2PO4
    137 mM NaCl
    4.16 mM NaHCO3
    0.34 mM NaH2PO4
    5 mM glucose
    5.36 mM sodium pyruvate
    5.36 mM HEPES
    0.001% phenol red
    Adjust the pH to 7.2 and filter the solution in a laminar flow with a vacuum filter system unit. Store at 4 °C, for no more than 1 month
  3. DNase I solution (5 mg/ml)
    5 mg DNase I in 1 ml of 0.15 M NaCl
    Store at -20 °C in small aliquots for 1 week
    Note: Higher concentrations of this solution may lose enzymatic activity.
  4. BSA solution (3 mg/ml)
    150 mg BSA in 50 ml HBSS
    In a laminar flow, filter the solution with a 0.20 μm acetate cellulose filter
    Prepare just before use
  5. Trypsin solution (0.5 mg/ml)
    2.5 mg trypsin in 5 ml BSA solution
    Add 50 μl of 5 mg/ml DNase I solution
    In a laminar flow filter the solution using a 0.20 μm acetate cellulose filter
    Prepare just before use
  6. Trypsin inhibitor solution (1 mg/ml)
    10 mg of trypsin inhibitor in 10 ml BSA solution
    In a laminar flow filter the solution with a 0.20 μm acetate cellulose filter
    Prepare just before use
  7. Washing solution
    Dilute the sterile trypsin inhibitor solution in BSA solution in a proportion of 1:5, to make a final volume of 10 ml
    Prepare just before use
  8. Inactivated FBS
    To inactivate the FBS stock solution, warm it in a water bath at 56 °C for 30 min
    Aliquot the FBS and store at -20 °C until shelf life expiration date
  9. Supplemented Neurobasal medium
    1. Supplement the 500 ml bottle of Neurobasal medium with 1.25 ml of L-glutamine and 0.5 ml of gentamicin (to obtain a final concentration of 0.5 mM and 50 μg/ml, respectively)
    2. Make aliquots of 49 ml Neurobasal medium in sterile centrifuge tubes
    3. Add 1 ml of B-27 supplement (50x) to achieve a final volume of 50 ml. Once supplemented, the Neurobasal medium is stable for up to 1 week if stored at 4 °C protected from light
    4. On the day of culture, and only for the plating medium, add 0.25 ml of inactivated FBS in 50 ml supplemented Neurobasal medium (for a final concentration of 0.5%)

Acknowledgments

We would like to thank Carina Maranga (CNC, University of Coimbra, Coimbra, Portugal) for assisting in filming brain dissection. This work was supported by European community FEDER fund through the ‘Programa Operacional Factores de Competitividade’ – COMPETE 2020; and national funds by ‘Fundação para a Ciência e a Tecnologia’ (FCT), Portugal [project ref. POCI-01-0145-FEDER-007440]. FVB/NJ mice used in these primary cultures were supported by a project financed by Teva Pharmaceutical Industries Ltd. The authors declare that they have no financial or competing conflict of interests.

References

  1. Graveland, G. A. and DiFiglia, M. (1985). The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum. Brain Res 327(1-2): 307-311.
  2. Hui, C. W., Zhang, Y. and Herrup, K. (2016). Non-neuronal cells are required to mediate the effects of neuroinflammation: results from a neuron-enriched culture system. PLoS One 11(1): e0147134.
  3. Matamales, M., Bertran-Gonzalez, J., Salomon, L., Degos, B., Deniau, J. M., Valjent, E., Herve, D. and Girault, J. A. (2009). Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice. PLoS One 4(3): e4770.
  4. Reiner, A., Albin, R. L., Anderson, K. D., D'Amato, C. J., Penney, J. B. and Young, A. B. (1988). Differential loss of striatal projection neurons in Huntington disease. Proc Natl Acad Sci U S A 85(15): 5733-5737.
  5. Welch, J. M., Lu, J., Rodriguiz, R. M., Trotta, N. C., Peca, J., Ding, J. D., Feliciano, C., Chen, M., Adams, J. P., Luo, J., Dudek, S. M., Weinberg, R. J., Calakos, N., Wetsel, W. C. and Feng, G. (2007). Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature 448(7156): 894-900.

简介

小鼠纹状体神经元的原代培养物被广泛用于探索神经生物学中的细胞机制,包括脑部疾病。 在这里,我们描述了一个详细和标准化的协议解剖和文化胚胎小鼠纹状体神经元GABA阳性/ DARPP-32阳性12天体外,当他们显示良好的神经元细胞连接性和存在的树突 刺,反映了网络的成熟。

【背景】纹状体是运动和奖励系统的重要组成部分,纹状体神经元的功能障碍可导致各种神经元疾病,从强迫性行为(Welch et al。,2007)到神经退行性疾病,如在亨廷顿病中观察到的(Reiner等,1988)。因此,一个良好建立的纹状体文化可能是一个很有价值的研究这些和其他条件的模型。成年纹状体中的主要神经元亚型是中型多刺投射神经元(MSNs),其占所有纹状体神经元的约95%并使用抑制性递质γ-氨基丁酸(GABA)。它们具有中等大小的细胞体,复杂的树突状乔木和高密度的树突棘,可同时接受谷氨酸能和多巴胺输入。其余5%的神经元由GABA能神经间神经元组成(Graveland和DiFiglia,1985; Matamales等人,2009)。以下方案描述了具有低百分比的星形胶质细胞的小鼠胚胎纹状体神经元的原代培养物的制备,其增殖被有丝分裂抑制剂5-氟-2'-脱氧尿苷(5-FdU)阻止。在体外12天后(DIV),通过所述方案获得的纹状体神经元预计将免疫标记微管相关蛋白2(MAP2)或NeuN,GABA,并且大部分用于多巴胺和cAMP调节的磷蛋白(DARPP-32)。

关键字:原代培养物, 纹状体解剖, 纹状体神经元, 中型多棘神经元

材料和试剂

  1. Coverslips直径为18mm,#1.5(0.17mm)厚(Thermo Fisher Scientific,Menzel,目录号:11817742)
  2. 无菌非组织培养处理的35,60和100mm的培养皿(VWR,目录号:734-1707,734-1708,734-1709)
  3. 5,10ml血清学无菌移液器(Labbox,目录号:MPIP-U05-200,MPIP-U10-200)
  4. 无菌0.2-10μl,10-200μl和100-1,000μl微量吸头(Frilabo,产品目录号:170558,171017,171024)
  5. 15和50ml无菌锥形离心管(Corning,目录号:430791,430829)
  6. 无菌1.5 ml微量离心管(BIOplastics,目录号:B74085)
  7. 无菌注射器,20毫升(Terumo,目录号:SS + 20L1)
  8. 无菌230毫米玻璃巴斯德移液器(Labbox,目录号:PIPN-230-250)
  9. 无菌0.2微米醋酸纤维素过滤器(GE Healthcare,Whatman,目录号:28415732)
  10. 真空0.2μm过滤瓶系统,500 ml(Corning,目录号:430769)
  11. 细胞过滤器,40微米(Corning,Falcon ,目录号:352340)
  12. 多孔组织培养处理的平底聚苯乙烯(Corning,目录号:3596,3548,3524,3512,3506)
  13. 妊娠16天的怀孕雌性小鼠( Mus musculus )
    注意:在此协议中,使用来自FVB / NJ近交系(THE JACKSON LABORATORY)的小鼠。
  14. 盐酸(HCl),37%(Sigma-Aldrich,目录号:435570)
  15. 乙醇绝对99.8%(Fisher Scientific,目录号:10342652)
  16. 异氟醚Iso-Vet 1000 mg / g吸入蒸气(Chanelle UK)
  17. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  18. 台盼蓝溶液,0.4%(Sigma-Aldrich,目录号:T8154)
  19. 5-FdU(Sigma-Aldrich,目录号:F0503)
  20. 抗MAP2(1:500; Santa Cruz Biotechnology,目录号:sc-32791)
  21. 抗GABA(1:500; Sigma-Aldrich,目录号:A2052)
  22. 抗DARPP32(1:100; Abcam,目录号:ab40801)
  23. 山羊抗兔IgG二抗Alexa Fluor 594(1:200; Thermo Fisher Scientific,Invitrogen,目录号:R37117)
  24. 驴抗小鼠IgG二级抗体Alexa Fluor 488(1:200; Thermo Fisher Scientific,Invitrogen,目录号:R37114)
  25. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  26. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P1149)
  27. 氯化钾(KCl)(Labbox,目录号:POCL-00P-1K0)
  28. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  29. 氯化钠(NaCl)(Merck,目录号:106404)
  30. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  31. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S3139)
  32. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  33. 丙酮酸钠(100mM)(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  34. HEPES(Sigma-Aldrich,目录号:H3375)
  35. 酚红(Sigma-Aldrich,目录号:P3532)
  36. 脱氧核糖核酸酶I(DNA酶I)(Sigma-Aldrich,目录号:D5025)
  37. 牛血清白蛋白(BSA)不含脂肪酸(Sigma-Aldrich,目录号:A6003)
  38. 来自猪胰腺的胰蛋白酶,IV-S型(Sigma-Aldrich,目录号:T0303)
  39. 胰蛋白酶抑制剂,II-S型:大豆(Sigma-Aldrich,目录号:T9128)
  40. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030081)。
  41. 庆大霉素(50 mg / ml)(Thermo Fisher Scientific,目录号:15750060)
  42. 神经基础培养基(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
  43. B-27补充剂(50x),无血清(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  44. 用于涂层的聚-D-赖氨酸(0.1mg / ml)(见食谱)
  45. Hanks的平衡盐溶液(HBSS)(见食谱)
  46. DNA酶I溶液(5毫克/毫升)(见食谱)
  47. BSA溶液(3毫克/毫升)(见食谱)
  48. 胰蛋白酶溶液(0.5毫克/毫升)(见食谱)
  49. 胰蛋白酶抑制剂溶液(1毫克/毫升)(见食谱)
  50. 洗涤液(见食谱)
  51. 失活的FBS(见食谱)
  52. 补充Neurobasal介质(见食谱)

设备


  1. 11.5厘米细直剪(Fine Science Tools,Dumont,目录号:14060-11)

  2. 8.5厘米微型解剖剪刀,直尖尖(世界精密仪器公司,产品目录编号:503669)
  3. 中型镊子(Fine Science Tools,型号:Dumont#7,目录号:11274-20)
  4. 镊子与直角和角度精尖(Fine Science Tools,型号:Dumont#5和Dumont#5/45)
  5. 自动移液器和微量移液器
  6. 玻璃肖特实验室瓶盖,500毫升(DWK生命科学,杜兰,目录号:21 801 44 5)
  7. 带电子温度调节的水槽
  8. 超声波清洗浴(仅用于盖玻片清洗)
  9. Zeiss Stemi放大镜(或同等产品)
  10. 相位倒置显微镜配备10倍和20倍物镜
  11. 蔡司LSM 710点扫描共焦显微镜(ZEISS,型号:LSM 710或同等产品)
  12. 血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  13. 水夹套CO2培养箱
  14. 垂直层流室
  15. 异氟醚蒸发器装置(E-Z麻醉,型号:EZ-SA800或同等产品)

软件

  1. 斐济软件(ImageJ,美国国立卫生研究院)

程序

注意:除了盖玻片清洗(在下面的程序A中描述,在聚-D-赖氨酸包被之前)和脑部剥离之外,所有的程序必须使用流动室在无菌环境中进行。

  1. 盖玻片清洗(时间约20小时;在聚-D-赖氨酸涂层之前)
    1. 将盖玻片放在一个松散覆盖的玻璃瓶中,在50-60℃下在1 M HCl中嵌入4-16小时,偶尔旋转。一次使用少量盖玻片以防止它们互相粘连。制造商推荐酸洗方案以增强盖玻片的清洁度并增加细胞附着。
    2. 在室温下冷却,小心地将HCl溶液倒入合适的容器中。用ddH 2 O彻底冲洗并用ddH 2 O填充玻璃瓶。在水浴中超声30分钟。重复超声波步骤两次。
    3. 用50%乙醇(v / v)溶液代替ddH 2 O,并在水浴中超声30分钟。用增加浓度的乙醇重复超声处理步骤两次:(i)70%乙醇(v / v),(ii)95%乙醇(v / v)。盖玻片可以储存在乙醇绝对≥99.8%(v / v)中长达6个月。
    4. 在使用前,对盖玻片进行消毒,使其暴露于紫外线下约15分钟或通过干热灭菌。

  2. 聚D-赖氨酸涂层(时间约2.5小时;培养前1天)
    1. 将预先制备的0.1mg / ml聚-D-赖氨酸溶液加入到培养所需的平板/盖玻片中,直到其覆盖底部。孵育板/盖玻片至少2小时,最好过夜,在37°C在潮湿的孵化器。
    2. 用巴斯德吸管吸出聚-D-赖氨酸并用无菌水冲洗两次。
      除去水,让井在层流室中干燥。
    3. 在4°C储存长达1个月。 

  3. 大脑和纹状体解剖(时间1小时30分钟)
    1. 在开始解剖之前,将所有解剖工具浸泡在70%乙醇(v / v)中。此外,在解剖过程中,常规将解剖工具浸入70%乙醇(v / v)中以减少污染。
      使用前彻底干燥
    2. 在妊娠第16天用5%异氟烷(v / v)麻醉怀孕的雌性小鼠,并通过颈椎脱臼处死它(或根据相应研究国家的机构动物护理和使用委员会裁决的建议选择适当的麻醉) 。将小鼠放在腹部,用70%乙醇(v / v)彻底冲洗腹部。通过剖宫产收集胚胎,用无菌镊子抓住子宫并切断与子宫内膜的连接。将胚胎放入装有HBSS的100毫米培养皿中。
    3. 从胚囊中取出每个胚胎并迅速将其去死。将胚胎的头部转移到HBSS填充的60毫米培养皿中,并保存在冰上。
    4. 使用一副镊子,握住胚胎的头部,并在眼睛之间插入一个直的Dumont#5镊子的尖端,以保持头部固定在表面之上。用微型解剖剪向上,开始从脊髓末端(在断头后可见)通过纵向裂隙切开皮肤和头骨,直到头部腔的末端。支持培养皿中的头部,并使用成角度的Dumont#5/45镊子,将脑部从脑腔中沿枕前方向轻轻挤出。
      每次将一个大脑转移到一个新的HBSS填充60毫米培养皿(见视频1)。

      视频1
      视频1.举例说明如何从头骨上移除鼠标大脑

    5. 打开放大镜并调整大约25倍的放大倍数。在大脑朝上的情况下,使用成角度的Dumont#5/45镊子,通过使平行于中线的矢状切割约1mm开始解剖。使用这个切口可以方便地将脑膜从皮质中去除(见视频2 /图1A-1D)。为了在解剖过程中稳定大脑,使用直的Dumont#5镊子轻轻地将大脑夹在前脑和小脑之间。

      视频2
      视频2.从胚胎E16小鼠脑中分离纹状体

    6. 用相同角度的钳子打开狭缝以展开覆盖侧脑室的前部皮层和神经节隆起。纹状体呈现灌注状的椭圆形结构,位于侧向隆起。如视频2 /图1E-1F所示,使用有角度的钳子的尖端来切除夹在四周的纹状体。转移到无菌HBSS填充35毫米培养皿。
    7. 重复另一个叶和每个大脑的程序,直到收集到所有的纹。可以利用剩余的组织分离海马,皮层或其他感兴趣的大脑区域。


      图1.胚胎E16小鼠大脑纹状体解剖的步骤A和B.背侧定位的胚胎大脑(C)平行于矢状缝的中央平面切割。用直的Dumont#5镊子夹住小脑(Ce)以在解剖过程中保持大脑稳定。 D.使用开放的缝隙拉出覆盖整个前脑(Fb)的脑膜(星号*)。之后,使用斜角Dumont#5/45镊子(E)展开前侧皮质(Cx)和(F)去除侧脑室旁边的纹状体(St),其具有典型的条纹外观,其不总是在放大25倍。 Ob,嗅球; B,脑干。

  4. 纹状体细胞的解离和电镀(时间30分钟)
    1. 将纹状体转移到无菌HBSS培养基(7毫升)中加入15毫升Falcon。
    2. 加入3毫升胰蛋白酶溶液,并轻轻翻转管混合。在37°C水浴中不时温和地混合2-3分钟。请注意,取决于组织的数量和胰蛋白酶活性,胰蛋白酶消化时间可能会改变。目前的方案优化到3毫升的胰蛋白酶解决方案约6胚胎;
      数量增加的胚胎可能需要调整溶液的总体积,维持胰蛋白酶的比例。
    3. 通过加入5ml胰蛋白酶抑制剂溶液来灭活胰蛋白酶。请注意,如果上一步添加的体积发生变化,则应相应添加胰蛋白酶抑制剂溶液的体积。轻轻翻转试管并等待组织沉降。吸引媒体。
    4. 通过加入5ml洗涤溶液(在BSA溶液中的胰蛋白酶抑制剂,1:5)洗涤细胞并通过翻转管混合。
      允许组织沉降并吸取尽可能多的体积,而不会干扰颗粒。
    5. 加入含有0.5%FBS的2-3ml补充的Neurobasal培养基,用P1000吸管轻轻匀浆,直至溶液均匀。请注意,如果胰蛋白酶消化步骤足够,则不需要进行剧烈研磨,因为机械均质过程中对细胞的太多物理压力会降低细胞活力。
    6. 用20μl细胞悬液,20μlNeurobasal培养基和20μl台盼蓝溶液制备1.5ml Eppendorf,产生3倍稀释因子。使用倒置相差显微镜计数血细胞计数器和平板中活细胞的数量,密度为130×10 3个细胞/ cm 2 2。对于盖玻片板中的低密度培养物92×10 3个细胞/ cm 2 2。将神经元在含有5%CO 2/95%空气的湿润培养箱中在37℃下维持12天。
    7. 在DIV3中取出三分之一的培养基,加入等体积的新鲜补充的不含FBS的Neurobasal培养基,并含有15μM5-FdU(最终浓度为5μM)以减少原代培养物中分裂的非神经元细胞(Hui 等。,2016)。在这些条件下,我们观察到约15%的胶质纤维酸性蛋白(GFAP)阳性细胞(数据未显示)。在DIV7中,通过将一半的培养基替换为不含FBS和5-FdU的新鲜补充的Neurobasal培养基来更换培养基。
    8. 根据神经元(MAP2或NeuN阳性),神经胶质细胞(GFAP或S100β阳性),GABA能细胞(GAD65或GABA阳性),MSNs(DARPP-32阳性和GAD65或GAD65)的百分比表征您的主要纹状体培养物。 GABA阳性)和GABA能中间神经元(GAD65阳性和DARPP-32阴性)。图2显示了具有大约82%的GABA阳性加DARPP-32阳性纹状体神经元的原代纹状体神经元培养物的代表性图像。 


      图2.小鼠原代纹状体培养的特征。 (A)将与DIV12培养的纹状体神经元一起用缀合有二级Alexa Fluor 594(1:200;实例:590nm,Em:617nm)的抗MAP2(1:500; Santa Cruz Biotechnology) (1:200;实例:495nm,Em:519nm)缀合的抗GABA(1:500; Sigma-Aldrich)或(B)抗DARPP32(1:100; Abcam)抗体。仅进行二次抗体对照染色,其不显示信号。在一个倒置共焦显微镜上用20倍物镜获得的Z-堆叠共焦图像,NA = 1.4。比例尺= 50微米。

数据分析

对于纹状体培养物表征,每个单独实验的6至8个图像随机用共聚焦显微镜针对标记的每种抗体进行。用Fiji软件(ImageJ,National Institute of Health,USA)和细胞计数器插件计算GABA-和DARPP-32-阳性细胞,并计算相对于MAP2阳性细胞的百分比。

食谱

  1. 用于涂层的聚-D-赖氨酸(0.1mg / ml)。
    1. 准备I型(超纯,> 17.4MΩ电阻)水中的166mM硼酸溶液并调节pH至8.2
    2. 使用前,稀释前一次制备的硼酸溶液中的聚-D-赖氨酸,并用过滤装置以层流过滤溶液。
  2. 汉克斯平衡盐溶液(HBSS)
    5.36 mM KCl
    0.44mM KH 2 PO 4 4 137mM NaCl
    4.16mM NaHCO 3 3 0.34mM NaH 2 PO 4 4 5 mM葡萄糖
    5.36 mM丙酮酸钠
    5.36 mM HEPES
    0.001%酚红
    调整pH值至7.2,并用真空过滤系统单元以层流过滤溶液。
    在4°C下储存不超过1个月
  3. DNase I溶液(5 mg / ml)
    5毫克脱氧核糖核酸酶I在1毫升的0.15 M NaCl中
    储存于-20°C小等分试样中1周 注:此溶液浓度较高可能会失去酶活性。
  4. BSA溶液(3毫克/毫升)
    150毫克BSA在50毫升HBSS
    在层流中,用0.20μm醋酸纤维素过滤器过滤溶液。
    在使用前准备
  5. 胰蛋白酶溶液(0.5毫克/毫升)
    2.5毫克胰蛋白酶在5毫升BSA溶液
    加入50μl的5 mg / ml DNase I溶液
    在层流过滤器中使用0.20微米醋酸纤维素过滤器的溶液
    在使用前准备
  6. 胰蛋白酶抑制剂溶液(1毫克/毫升)

    10毫克胰蛋白酶抑制剂在10毫升BSA溶液中 在层流过滤器中使用0.20μm醋酸纤维素过滤器的溶液 在使用前准备
  7. 洗涤解决方案
    按1:5的比例稀释BSA溶液中的无菌胰蛋白酶抑制剂溶液,使最终体积为10 ml
    在使用前准备
  8. 未激活的FBS
    为了灭活FBS储备溶液,在56℃的水浴中加热30分钟
    分装FBS并在-20°C储存直至保质期到期日
  9. 补充的Neurobasal培养基
    1. 用1.25ml L-谷氨酰胺和0.5ml庆大霉素补充500ml的Neurobasal培养基(分别获得终浓度为0.5mM和50μg/ ml)

    2. 在无菌离心管中分装49 ml Neurobasal培养基
    3. 加入1毫升B-27补充剂(50x)以达到最终体积50毫升。补充后,如果储存在4°C避光条件下,Neurobasal培养基可稳定长达1周
    4. 在培养当天,只有电镀培养基中,加入0.25ml灭活的FBS在50ml补充的Neurobasal培养基中(终浓度为0.5%)。

致谢

我们要感谢Carina Maranga(CNC,科英布拉大学,科英布拉,葡萄牙)协助拍摄脑部清扫。这项工作得到了欧洲共同体FEDER基金通过“竞赛运动竞赛案例” - COMPETE 2020的支持;和国家基金,由葡萄牙的“Fundaçãopara aCiênciae a Teologia”(FCT)[项目编号。 POCI-01-0145-FEDER-007440。在这些原始培养物中使用的FVB / NJ小鼠得到了由Teva制药工业有限公司资助的项目的支持。作者宣称他们没有财务或竞争利益冲突。

参考

  1. Graveland,G.A。和DiFiglia,M.(1985)。 灵长类动物和啮齿动物新纹状体中具有缩进核的中等大小神经元的频率和分布。 a> Brain Res 327(1-2):307-311。
  2. Hui,C. W.,Zhang,Y.和Herrup,K。(2016)。 需要非神经细胞来介导神经炎症的效应:神经元富集培养系统的结果。 PLoS One 11(1):e0147134。
  3. Matamales,M.,Bertran-Gonzalez,J.,Salomon,L.,Degos,B.,Deniau,J.M.,Valjent,E.,Herve,D.and Girault,J.A。(2009)。 纹状体中型棘状神经元:核染色鉴定和BAC转基因小鼠中神经元亚群的研究。 PLoS One 4(3):e4770。
  4. Reiner,A.,Albin,R.L.,Anderson,K.D。,D'Amato,C.J.,Penney,J.B。和Young,A.B.(1988)。 亨廷顿病中纹状体投射神经元的微量损失 ProcNatlAcadSci USA 85(15):5733-5737。
  5. Welch,JM,Lu,J.,Rodriguiz,RM,Trotta,NC,Peca,J.,Ding,JD,Feliciano,C.,Chen,M.,Adams,JP,Luo,J.,Dudek,SM,Weinberg ,RJ,Calakos,N.,Wetsel,WC和Feng,G。(2007)。 Sapap3突变小鼠的皮质纹状体突触缺陷和OCD样行为 Nature 448(7156):894-900。
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引用:Naia, L. and Rego, A. (2018). Isolation and Maintenance of Striatal Neurons. Bio-protocol 8(8): e2823. DOI: 10.21769/BioProtoc.2823.
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