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Aug 2019

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Generation of Functional Mouse Hippocampal Neurons
功能性小鼠海马神经元的产生   

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Abstract

Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos’ isolation, culturing conditions and assessment of culture’s purity and differentiation. Evaluation of neuronal activity was performed by analysis of calcium imaging dynamics at six days in culture.

Keywords: Primary cultures (原代培养), Hippocampal neurons (海马神经元), Hippocampus isolation (海马分离), Primary neurons (原代神经元), Mouse (小鼠), Neuronal calcium imaging (神经元钙成像)

Background

The hippocampus is a very well-characterized brain structure critical for important cerebral functions such as memory, spatial navigation, emotional memory and learning. Anatomically, the murine hippocampus has a well-defined, C-shaped structure that is easy to locate and isolate. At the cellular level, it is mainly composed of pyramidal cells with fewer interneurons and glial cells compared to other brain regions (Kaech and Banker, 2006). As such, the hippocampus is an ideal region for generating primary neuron cultures of high purity from wild-type or genetically engineered mouse models that can be used for disease modeling or to investigate multiple aspects of neuronal function such as synaptic transmission and electrophysiological properties, sensitivity to neurotoxicity, differentiation and aging (Busche, 2018; Koyama and Ikegaya, 2018; Molnar, 2011; Wu et al., 2019; Rush et al., 2020).

Many protocols have been developed for generating cortical and hippocampal neuron cultures by co-culturing neurons with glial feeders (Kaech and Banker, 2006) describing three-dimensional neuronal culture systems with astrocytes encapsulated in hydrogel microfibers (Kim et al., 2020), supplementing culture medium with growth factors for long-term neuron cultures (Ray et al., 1993), or generating neurons from post-natal mice (Beaudoin et al., 2012). Each of these methodologies presents advantages and disadvantages depending on the goals of the experiment. Here, we extend and describe in detail a protocol for generating hippocampal neurons that we have developed and successfully employed over the years (e.g., Dorsey et al., 2006; Tomassoni-Ardori et al., 2019). We focus primarily on providing an easy, simple and reliable procedure for obtaining primary hippocampal neurons from mice at the late embryonic stage (E17.5-E18.5). We illustrate the whole preparation process including aspects that have often been omitted in other protocols such as a description of dissection instruments and surgical procedures for embryos removal and dissection. We report analysis of neuronal differentiation and function after 6 days in culture by testing for neuronal differentiation markers and neuronal activity by cellular calcium imaging dynamics. Moreover, we show the levels of neuronal culture purity in relation to glial cells with and without treatment with the glial inhibitor cytosine arabinoside (Ara-C).

This step-by-step protocol allows the experimenter to obtain differentiated hippocampal neurons with a high degree of purity within 6 days.

Materials and Reagents

The following list provides examples of the materials and equipment that we routinely use in our laboratory. Reagents and equipment with similar specifications will work as well.

  1. Poly-D-lysine/laminin cellware 12 mm round coverslips (Corning, catalog number: 354087 )
  2. Sterile glass Pasteur pipettes (Thomas Scientific, catalog number: 1215D99 )
  3. 24-well plate (Corning, catalog number: 3524 )
  4. 35 mm TC-treated culture dish (Corning, catalog number: 353001 )
  5. 100 mm TC-treated culture dish (Corning, catalog number: 430167 )
  6. Cellometer disposable counting slides (SD100 slides) (Nexcelom Bioscience, catalog number: CHT4-SD100-002 )
  7. 50 ml conical tube (Corning, catalog number: 430828 )
  8. 15 ml conical tube (Corning, catalog number: 430790 )
  9. InvitrolonTM PVDF/Filter Paper Sandwiches (for mini gels) (Thermo Fisher Scientific, catalog number: LC2005 )
  10. E17.5/E18.5 mouse embryos
  11. Neurobasal medium (Thermo Fisher Scientific, catalog number: 21103-049 )
  12. B27 supplement (Thermo Fisher Scientific, catalog number: 17504-044 )
  13. Dulbecco's modified high glucose eagle medium (DMEM) (Thermo Fisher Scientific, catalog number: 11965-092 )
    Note: This DMEM formulation contains 4500 mg/L glucose, L-glutamine and phenol red.
  14. Distilled water (Thermo Fisher Scientific, catalog number: 15230-162 )
  15. Poly-D-lysine (Sigma Aldrich, catalog number: P6407 )
  16. Fetal bovine serum (not heat-inactivated) (Omega Scientific, catalog number: FB-01 ; or other brand FBS)
  17. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200-056 )
  18. Penicillin/Streptomycin (P/S) 10,000 U/ml (100x concentrated) (Thermo Fisher Scientific, catalog number: 15140-122 )
  19. Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190-144 )
  20. Trypan blue (Sigma-Aldrich, catalog number: 93595 )
  21. Cytosine arabinoside (Ara-C) (Sigma-Aldrich, catalog number: C6645 )
    Note: Stock solution is reconstituted in water at a concentration of 50 mg/ml, aliquoted and stored at -20 °C.
  22. Anti-Tubulin-β-III antibody (mouse monoclonal) (Covance, catalog number: MMS-435P )
  23. Anti-GFAP antibody (rabbit polyclonal) (Agilent (Dako), catalog number: Z0334 )
  24. Alexa FluorTM 488 donkey anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, catalog number: A-21206 )
  25. Alexa FluorTM 647 donkey anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, catalog number: A-31571 )
  26. DAPI (Invitrogen, catalog number: D1306 )
  27. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  28. TritonTM-X100 (Sigma-Aldrich, catalog number: T8532 )
  29. Donkey serum (Sigma-Aldrich, catalog number: S30-100ML )
  30. Bovine serum Albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
  31. Fluorescence mounting medium (Dako, catalog number: S3023 )
  32. Ethanol solution 70%
  33. Laminin Mouse Protein, Natural (Thermo Fisher Scientific, catalog number: 23017-015 )
  34. Sample buffer, Laemmli 2x concentrate (Sigma-Aldrich, catalog number: S3401 )
  35. NuPAGE 4 to 12%, Bis-Tris, Mini Protein Gel (Thermo Fisher Scientific, catalog number: NP0321PK2 )
  36. Tween 20 (Sigma Aldrich, catalog number: P1379 )
  37. Non-fat dry milk (Santa Cruz Biotechnology, catalog number: sc-2324 )
  38. Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific, catalog number: 34076 )
  39. Anti-TrkB antibody (Millipore, catalog number: 07-225 )
  40. Anti-Rbfox1 antibody clone 1D10 (Millipore, catalog number: MABE985 )
  41. Anti-Rbfox3 (NeuN) antibody (Millipore, catalog number: MAB377 )
  42. Anti-mouse IgG, HRP-conjugated (Millipore, catalog number: AP192P )
  43. Anti-rabbit IgG, HRP-conjugated (Millipore, catalog number: AP182P )
  44. Fluo-4 DMSO solution 1 mM (Thermo Fisher Scientific, catalog number: F14217 )
  45. PowerLoad Concentrate 100x (Thermo Fisher Scientific, catalog number: P10020 )
  46. Probenecid water soluble (Thermo Fisher Scientific, catalog number: P36400 )

Equipment

  1. Dissection hood (NuAire, model: NU-201-430 )
  2. Laminar flow hood (NuAire, model: NU-425-400 , Class II, Type A/B3)
  3. Stereoscopic microscope (Olympus, model: SZH-ILLK )
  4. Inverted tissue culture microscope (Nikon, model: Diaphot )
  5. CO2 incubator for cell culture (Sanyo, model: MCO-17AIC )
  6. Surgical scissors, sharp (Fine Science Tools, catalog number: 14002-16 )
  7. Student standard pattern forceps (Fine Science Tools, catalog number: 91100-12 )
  8. Fine scissors, sharp (Fine Science Tools, catalog number: 14060-10 )
  9. Graefe forceps (Fine Science Tools, catalog number: 11052-10 )
  10. Dumont #7 forceps (Fine Science Tools, catalog number: 11272-30 )
  11. Cell counter (Nexcelom Bioscience, Cellometer Vision)
  12. Centrifuge (Thermo Scientific, model: CL2 )
  13. Tissue culture hood (NuAire, model: NU-425-400 )
  14. Confocal microscope system (Zeiss, model: LSM780 )
  15. Bunsen burner (HanauTM Touch-O-Matic)
  16. Branson 1800 sonicator (Branson)
  17. XCell SureLock Mini-Cell protein electrophoresis (Thermo Fisher Scientific, catalog number: EI0001 )
  18. GeneGnome XRQ-Chemiluminescence Imaging system (Syngene)

Procedure

Notes:

  1. This methodology describes the critical steps necessary for generating primary hippocampal neurons derived from E17.5/E18.5 mouse embryos. Practical tips after each step are included to better guide the readers through the procedure.
  2. Depending on the final experimental aims, different tissue culture dishes/plates or glass coverslips can be used for culturing primary hippocampal neurons.

  1. Coating of tissue culture dishes/plates with poly-D-lysine
    1. Reconstitute poly-D-lysine in a sterile laminar hood by adding 50 ml of sterile tissue culture grade water to a 5 mg γ-irradiated and cell culture tested poly-D-lysine bottle (final working concentration of 0.1 mg/ml).
    2. Evenly coat the culture dish surface with 0.1 mg/ml poly-D-lysine solution and incubate at room temperature for 20 min in a laminar flow hood.
    3. Remove the poly-D-lysine solution by aspiration and rinse the surface thoroughly with sterile tissue-culture grade water twice.
    4. Before plating the cells, allow the surface to dry completely by leaving the dishes/plates uncovered under the laminar flow hood for about 1 h.
      Note: If plating cells on glass coverslips use commercially available poly-D-lysine/laminin pre-coated 12 mm coverslips. Transfer poly-D-lysine/laminin coated coverslips directly into wells of a 24-well plate by using sterile forceps in a sterile laminar flow hood before plating neurons. Alternatively, regular 12 mm coverslips can be coated by placing them into 24-well plate wells and covering them with 0.1 mg/ml poly-D-lysine for 20 min in a laminar flow hood. After that wash twice with PBS and cover with a 10 µg/ml laminin solution for at least 4 h at 37 °C by leaving the coverslips in an incubator until cells are ready to be plated. Rinse twice with PBS just before plating the cell to prevent the culture surface from drying. The 1 mg/ml laminin stock solution is made by adding 1 ml of PBS into 1 mg laminin powder and diluting to 10 µg/ml laminin with PBS. Coating of coverslips in plates avoids the potential to scrape the coating off while transferring between dishes.

  2. Sacrifice pregnant mouse and remove E17.5/E18.5 embryos
    1. Euthanize pregnant mouse according to Animal Care and Use Committees (ACUC) approved protocols (CO2 euthanasia or isoflurane anesthesia followed by decapitation or cervical dislocation).
    2. Pin the animal to a clean surgical area and use 70% ethanol to clean the abdomen.
    3. In order to expose the embryos, perform an incision in the middle of the abdomen using surgical scissors (Figure 1A) starting from the pubic region and continuing up to the end of the abdominal cavity as indicated by the dashed line in Figure 2A.
      Note: Use forceps (Figure 1B) to pinch and pull up the skin layer while performing the incision with surgical scissors. This will help to avoid accidental damage to the embryos located immediately underneath.


      Figure 1. Dissection instruments used for the preparation of hippocampal neurons. A. Surgical scissors used in the protocol Steps B3-B6. B. Standard pattern forceps used in the protocol Steps B3-B6 and C1. C. Fine scissors used in Step C1. D. Curved forceps (Graefe forceps) used in Steps C2-C3. E. Dumont forceps used in Steps C4 and C5.


      Figure 2. Preparation of E17.5/18.5 pregnant animal for surgical removal of the embryos. A. Pinned animal showing with a dashed white line the location of the incision (Step B3); B. Exposure of uterus after abdominal incision (Step B3).

    4. Carefully expose the whole uterus (uterine horns) containing the embryos (Figure 2B) by using forceps and surgical scissors (Figures 1A-1B) and immediately transfer the uterus to a 100 mm dish containing ice-cold PBS (Figure 3A).
      Note: Use forceps to grab and pull the uterus out of the abdomen while using surgical scissors with the other hand to cut any adherence and the cervix connection (Figure 2B arrows).
    5. Continue to use forceps and surgical scissors to free the single embryos and carefully remove the placenta and yolk sack. Transfer the embryos into a new 100 mm dish containing ice-cold PBS to wash embryos and to remove blood and embryonal fluids (Figure 3B).
    6. Use forceps (Figure 1B) to hold the embryo and cut the head using surgical scissors (Figure 3C). Transfer the heads into a new 100 mm dish containing ice-cold DMEM complete with 10% fetal bovine serum and 1% antibiotics (P/S).
      Note: From this step forward, it is important to keep transferring the biological material to new dishes with clean medium containing antibiotics to lower the possibility of contamination. In the figures shown in this protocol PBS was intentionally used instead of medium containing phenol red only for the purpose of obtaining more clear images (Figure 3A, Figure 4, Figure 5 and Figure 6).


      Figure 3. Isolation of the embryos from the uterus. A. Isolated uterus (Step B4); B. Isolated mouse embryos (Step B5); C. Embryo heads (Step B6). D. Side view of embryo’s head. Dashed white line indicates where to execute the transversal cut described in Step C1 starting from the back of the skull (black scissors) while holding the embryo’s head with forceps in the nose area (black arrow).

  3. Dissection of hippocampi
    1. Move to a dissection cabinet with sterile laminar air flow and prepare a 100 mm dish containing ice-cold complete DMEM with 10% fetal bovine serum and 1% antibiotics (P/S) for dissection of the brains. Transfer one embryo head at a time into the 100 mm dish containing complete DMEM medium and gently remove the brain from the embryo head (Figure 4A). Specifically, with one hand, use forceps (Figure 1B) to pinch the mouth/nose area of the head (black arrow in Figure 3D). With the other hand, use the fine scissors (Figure 1C) to enter from the back of the skull (black scissors in Figure 3D) and execute a transversal cut (along the transversal plane at the mouth/nose level indicated by the white dashed line in Figure 3D) in order to free the basal brain region. Invert the embryo head to expose the free basal area of the brain. Continue by using two forceps (Figure 1B) to gently pull apart the skull and carefully remove the whole brain (Figure 4A). Transfer and collect the brains into a new 100 mm dish containing clean, ice-cold complete DMEM medium. Replace the medium after each brain dissection to eliminate the blood.
    2. Transfer one brain at a time to a 35 mm dish lid filled with ice-cold complete DMEM medium and move under a dissection stereoscope.
      Note: The lower depth of the lid of a 35 mm dish allows for better movements when using dissection instruments compared to the bottom of the 35 mm dish.
    3. Use two curved forceps (Figure 1D): hold the brain on the side with one forceps (white dots in Figure 4A) and insert the second forceps along the midline (dashed line in Figure 4A) to separate the two hemispheres of the brain. Continue to use curved forceps to gently pull apart and flip the two halves of the brain in order to expose and visualize the hippocampal area (Figures 4B-4C).
      Note: Due to the fragility and softness of the embryonal tissues, the brain can be easily damaged while trying to dissect the hippocampal region: always use care when handling and holding embryonal brains. A few practicing sessions at the stereoscope will help to improve dissecting skills quickly.


      Figure 4. Localization of the hippocampus. A. Dissected embryo brain. Dashed white line indicates where to separate the two brain hemispheres using one curved forceps while holding the brain on the side by using the second curved forceps (white dots) (Step C3). B. Separated brain hemispheres. White arrow heads indicate the location of the hippocampal region on each hemisphere (Step C3). C. Higher power image showing the hippocampal region on one brain hemisphere indicated by a white dashed line.

    4. Carefully remove the meninges from each brain hemisphere (Figure 5).
      Notes:
      1. Work by using two Dumont forceps (Figure 1E). Use one forceps to hold the whole brain and use the second forceps to gently peel the meninges off. Start by pinching the olfactory bulb area and gradually pulling back in order to remove meninges completely (Figure 5). Hold the brain gently while performing this operation and it might be necessary to change the holding points many times according to how easily the meninges come off.
      2. It is important to properly remove the meninges to eliminate as much non-neuronal cell contribution to the neuronal culture as possible.


      Figure 5. Removal of the meninges. Arrows indicate hippocampal and cortical regions as well as olfactory bulb and meninges removed from one brain hemisphere (Step C4).

    5. Proceed by using Dumont forceps to finely cut along the hippocampus edges to completely free the hippocampal area (Figures 5-6). Use curved forceps to gently pick and transfer each hippocampus to a new 35 mm tissue culture dish containing 2 ml of ice-cold serum-free DMEM medium with 1% antibiotics (P/S) and keep it on ice.
      Note: The hippocampus is distinguishable by its typical C-shaped anatomical structure and by a difference in light contrast under a stereoscope compared to the cortical area to which it is connected (Figures 5-6). After removing the hippocampus it is possible to collect cortices from the same embryos for the preparation of cortical neurons if needed (not described in this protocol).

  4. Trypsinization of the hippocampi
    1. Mince the hippocampal tissues in the same 35 mm tissue culture dish containing 2 ml of ice-cold serum-free DMEM medium used for the collection by cutting them into small pieces using two Dumont forceps (ideally 5-6 pieces from each hippocampus of approximately 2-3 mm3) (Figure 6C).
      Note: It is important to collect and keep the hippocampi in serum-free DMEM medium (Steps C5 and D1) to avoid inhibition of Trypsin-EDTA activity during the next step (Step D2).


      Figure 6. Isolation of the hippocampus (Step C5 and Step D1). A. Isolated hippocampi from one embryonal brain. B. higher power image of the hippocampal region showing the typical C-shaped anatomical structure. C. Minced hippocampi. Hippocampal tissue-chunks generated for trypsin digestion (Step D1).

    2. Move to a tissue culture hood with sterile laminar air flow and use a pipette to transfer the medium containing the minced hippocampi to a 50 ml sterile conical tube. Gently add 2 ml of 0.25% Trypsin-EDTA to the hippocampi and place the tube in a water bath at 37 °C for 25 min (final trypsin concentration of 0.125%).
      Note: During the 25 min digestion in trypsin, the minced hippocampal pieces will naturally sediment by gravity to the bottom of the 50 ml conical tube.
    3. Prepare a 15 ml sterile conical tube containing 2 ml of warm (37 °C) DMEM complete with 10% fetal bovine serum and 1% antibiotics (P/S). At the end of the digestion, carefully transfer the 50 ml digestion tube back to a tissue culture hood without disturbing or resuspending the hippocampal sediment and, with a pipette with a 1 ml tip, transfer only the hippocampal sediment to the new 15 ml conical tube containing the 2 ml of complete medium.
      Note: Use the 1 ml tip to directly contact the hippocampal sediment at the bottom of the 50 ml tube. Gently aspirate and transfer only the hippocampi at the bottom of the tube without excess liquid from the digestion. The fetal bovine serum contained in the new 2 ml medium will inactivate the trypsin and stop its enzymatic activity.

  5. Trituration of the hippocampal tissue
    1. Use a Bunsen burner to fire-polish a sterile glass Pasteur pipette and generate a smaller opening and a smoothened tip for dissociating the hippocampal tissue (Figure 7). Use the fire-polished pipette shown in Figure 7B to slowly and gently triturate the hippocampal pieces. Pipet up and down six to eight times.
      Note: Trituration of the hippocampi is a critical step to achieve good cell yield and cell viability. After fire-polishing, be sure to let the glass pipette cool down before triturating the hippocampi. Hippocampal chunks should be completely broken up after pipetting up and down six to eight times.
    2. Use the Bunsen burner again to prepare a new fire-pulled glass Pasteur pipette with a longer and tapered tip (Figure 7C). Use this pipette to slowly perform a single up and down pipetting of the hippocampal cell slurry in order to release single cells. After obtaining a single cell suspension proceed immediately to Procedure F. Keep the single-cell suspension in a tissue culture hood with sterile laminar air flow at room temperature while counting the cells.
      Note: Performing multiple pipetting with the fire-pulled Pasteur pipette with a longer and tapered tip (Figure 7C) will damage the cells and affect cell viability.


      Figure 7. Glass Pasteur pipette tips (Steps E1 and E2). A. Intact glass Pasteur pipette tip. B. Fire-polished glass Pasteur pipette with smoothened and smaller tip. C. Fire-polished glass Pasteur pipette with tapered and extra-small tip.

  6. Cell counting, cell seeding and cell culture
    1. Collect 50 μl of the cell suspension and add 5 μl of Trypan blue dye to assess cell viability. Count cells using a cell counter.
      Note: Cell viability ranges between 70-90% of live cells. As an example, the cell viability and cell count of a neuronal preparation from 8 embryos is about 4 x 106 live cells from a total of 5 x 106 cells (cell viability = 80%).
    2. Centrifuge the remaining hippocampal cell suspension at 350 x g for 5 min. Discard the supernatant and resuspend the cell pellet by pipetting gently up and down in warm Neurobasal medium with B27 supplement. Adjust the volume of medium according to the tissue culture plates that will be used. Use a seeding density of approximately 260,000 cells/square centimeter. Seed the cells in the tissue culture dish/plate previously coated with poly-D-lysine.
      Note: Cell yield is approximately 400,000-600,000 cells/embryo. Prepare complete medium for neuronal culture by adding 10 ml of B27 supplement and 5 ml of P/S antibiotics (100x concentrate) into 500 ml of Neurobasal medium. Some protocols (e.g., Ruhl et al., 2019) suggest to pre-plate neurons for one hour in DMEM medium with 10% FBS to facilitate neuron adhesion to the plate. However, we have found that plating directly in Neurobasal medium with B27 supplement is sufficient for good neuron adhesion.
    3. After 18 to 24 h from the initial plating, add cytosine arabinoside (Ara-C) to the culture to stop the proliferation of dividing cells (non-neuronal cells). Remove half of the volume of the culture medium (Neurobasal medium containing B27 supplement) and replace it with fresh medium containing 2 µM Ara-C (1 µM Ara-C final concentration).
      Note: Failing to add Ara-C to a primary neuron culture will considerably affect the purity of the culture (Figures 8B and 8F). Death of some mitotic cells can be observed after adding Ara-C to the neuron culture.
    4. Culture the primary hippocampal neurons by replacing only half of the medium every 48 to 72 h. It is not necessary to continue adding Ara-C when replacing medium after the initial treatment. After 6 days in culture, the primary neurons will be well developed and differentiated as assessed by both morphology but especially expression of typical neuronal markers such as Tubulin-β-III, NeuN, Rbfox1, neurotrophin receptors and several other neuronal proteins (Tomassoni-Ardori et al., 2019) (Figure 8 and Figure 9).
      Notes:
      1. Replacing only half of the culture medium maintains the growth factors naturally secreted and present in the neuronal conditioned medium.
      2. Although absolute prevention of microbial contamination (bacteria, mycoplasma, fungi) during the preparation of primary cell cultures is impossible, these steps can greatly improve the outcome: i) work in a sterile environment; ii) use proper aseptic technique including wearing sterile gloves and using sterile glassware and disposable pipettes and tips; iii) keep all the equipment clean (dissecting microscope, incubators, centrifuges and water baths).


      Figure 8. Development and differentiation of neurons in culture. A. Brightfield images of primary hippocampal neuron cultures from day 1 to day 6 in vitro. Note the increasing complexity of the neuronal network over time in vitro (magnification 20x). B. Western blot analysis of hippocampal neurons after 6 days in vitro. Immunoblots were performed with antibodies against markers of differentiated neurons: the BDNF receptor TrkB extracellular domain to detect all TrkB isoforms, Rbfox1, beta-III-tubulin (b-III-Tub) and Rbfox3 (NeuN). Molecular Weight markers are indicated on the left.

  7. Western blot analysis for the detection of markers of differentiated neurons (Figure 8B)
    1. After 6 days of in vitro culture, remove the culture medium from one well of a 24-well plate containing neurons.
    2. Rinse cells with PBS once.
    3. Lyse cells by adding 150 µl of 2x Laemmli sample buffer directly into the well.
    4. Transfer the cell lysate to a 1.5 ml tube and sonicate the sample for 10 min to shred genomic DNA and eliminate viscosity.
    5. Heat the sample at 95°C for 5 min to promote protein denaturation.
    6. Load 10 µl of sample per lane into a 4-12% NuPAGE pre-cast gel and run the gel at 120 V.
    7. Transfer gel to a PVDF membrane.
    8. Block membrane with 5% non-fat milk in TBS-Tween (0.1%) for 1 h at room temperature.
    9. Incubate membrane overnight with specific primary antibodies at 4 °C while gently shaking. Dilute primary antibodies 1:1,000 in 5% non-fat milk in TBS-Tween (0.1%) buffer.
    10. Wash membrane three times for 5 min with TBS-Tween (0.1%) buffer.
    11. Incubate membrane with HRP-conjugated secondary antibodies at room temperature for 1 h while gently shaking. Dilute secondary antibodies 1:5,000 in 5% non-fat milk in TBS-Tween (0.1%) buffer.
    12. Wash membrane three times for 5 min with TBS-Tween (0.1%) buffer.
    13. Incubate membrane with enhanced chemiluminescence (ECL) substrate and acquire images with a chemiluminescence imaging system or by film exposure.

  8. Phenotypic analysis of hippocampal neurons by immunofluorescence (Figure 9)
    1. After 6 days in culture, remove the culture medium from the 24-well plate containing the poly-D-lysine/laminin pre-coated 12 mm coverslips with hippocampal neurons. Rinse the cells with PBS and fix the cells with a 4% paraformaldehyde/PBS solution for 30 min at 4 °C.
    2. Rinse cells with PBS calcium/magnesium free (PBS -/-) 3 times for 5 min.
    3. Use a blocking solution for 1 h at room temperature.
      Note: Blocking solution is prepared using 10% normal donkey serum (v/v), 0.1% TritonTM-X100 (v/v), and 0.1% BSA (w/v) in PBS -/-.
    4. Incubate the cells with specific primary antibodies overnight at 4 °C.
      Note: Dilute primary antibody in a solution containing 1% normal donkey serum, 0.1% TritonTM-X100 (v/v), and 0.1% BSA (w/v) in PBS -/-. The dilution of anti-GFAP antibody is 1:1,000 and the dilution of anti-Tubulin-β-III antibody is 1:200 (see Materials and Reagents).
    5. Rinse the cells with PBS -/-, 3 times for 5 min.
    6. Incubate the cells with a specific secondary antibody and DAPI staining for nucleus visualization (0.5 µg/ml) for 2 h at room temperature. Alexa Fluor 488 Donkey anti-rabbit IgG (H+L): secondary antibody for anti-GFAP antibody; Alexa Fluor 647 Donkey anti-mouse IgG (H+L): secondary antibody for anti-Tubulin-β-III antibody. Dilute secondary antibodies 1:250 using the same solution as in Step G4.
    7. Rinse the cells with PBS -/-, 3 times for 5 min.
    8. Invert coverslips and mount them to a glass slide using Dako fluorescence mounting medium. After the mounting medium is dry the samples can be analyzed by confocal imaging.
      Note: Although in this protocol we show that differentiated hippocampal neurons can be obtained by 6 days in culture and we recommend the use of Ara-C for improving neuronal culture purity (Figure 8 and Figure 9), in some cases co-culturing neurons with glial cells can further support neuronal maturation and long-term survival (Kaech and Banker, 2006; Kim et al., 2020).


      Figure 9. Immunofluorescence staining of primary hippocampal neurons after 6 days in culture (Procedure G). Neurons were cultured with (A-D) or without (E-H) cytosine arabinoside (Ara-C 1 µM) and stained for the glial cell marker Glial Fibrillar Acidic Protein (GFAP in green), the neuronal marker beta-III-tubulin (b-III-TUB in red) and the nuclei dye (DAPI in blue). Note the significant number of GFAP-positive cells in the neuron culture without Ara-C treatment (F) after 6 days. Higher magnification images (I-K) of neurons cultured with Ara-C 1 µM and stained for beta-III-tubulin (b-III-TUB in green) and the nuclei dye (DAPI in blue). Note the neuronal-network complexity after 6 days in vitro.

  9. Functional phenotypic characterization of hippocampal neurons by calcium imaging (Figure 10 and Video 1)
    A further test of functionally differentiated neurons includes imaging of the calcium dynamic occurring in vitro following spontaneous or glutamate-induced depolarization (Grienberger and Konnerth, 2012)
    1. Preparation of Fluo-4 loading buffer: to prepare 1 ml of loading buffer add 1 µl of stock Fluo-4 (Fluo-4 stock: 1 mM in DMSO; final concentration 1 µm) in 10 µl of PowerLoad (100x concentrate). Then add 10 µl of Probenecid (stock solution 250 mM; final concentration 2.5 mM) and 979 µl of ECS buffer (140 mM NaCl, 4.7 mM KCl, 2 mM NaHCO3, 1 mM NaH2PO4, 1.2 mM MgCl2, 1.5 mM CaCl2, 3 mM glucose and 10 mM HEPES, pH 7.4).
    2. After 6 days in vitro, remove the culture medium from the 24-well plate with the coverslips containing the hippocampal neurons. Incubate each coverslip with 250 µl of Fluo-4 loading buffer at 37 °C for 30 min following by a 30 min wash in ECS buffer at 37 °C in order to allow the de-esterification and activation of Fluo-4.
    3. Adjust the flow in the recording chamber to 0.5 ml x min and set the temperature to 37 °C.
    4. Place the coverslip in the recording chamber and select a suitable field.
    5. Set focus, set laser intensity (480 nm) at a lower possible value and integration time (pixel dwell time) such that the acquisition time is shorter than 500 ms before starting the time series acquisition at the confocal at 2 images x second.
    6. Record neuronal spontaneous activity and then apply glutamate at 1 mM for 10 s to depolarize and visualize all the cells in the field.
    7. For image analysis import the file in ImageJ (or Fiji https://imagej.net/Fiji/Downloads); set the lookup table to Rainbow RGB; adjust image brightness and contrast; open the ROI manager (Analyze/Tools/ROI manager); select an appropriate area shape to cover a neuron of interest and then select Images/Stacks/Plot Z-axis Profile to plot the photon density of the selected ROI over time. Add the ROI to the ROI manager and proceed with another ROI. Density plot can be exported to a spreadsheet for further quantification.


      Figure 10. Calcium imaging of primary hippocampal neurons after 6 days in vitro (Procedure I). Single neuron traces of calcium fluorescence intensity over time of primary hippocampal neurons loaded with the calcium indicator Fluo-4 (A). Note the spontaneous activity of neurons before treatment with 1 mM glutamate (blue bar). Single neurons analyzed in (A) are indicated with numbers in (B). Representative images of calcium fluorescence in the neuronal culture before (C) or after (D) stimulation with 1 mM glutamate.

      Video 1. Calcium live imaging of primary hippocampal neurons after 6 days in vitro (Figure 10; Procedure I)

Acknowledgments

We thank Eileen Southon and Andie Kealohi Sato Conching for critical reading of the manuscript. This work was supported by the NIH Intramural Research Program, Center for Cancer Research, National Cancer Institute.

Competing interests

The authors declare no competing interests and mentioning of specific materials, reagents and equipment does not imply endorsement by the National Institutes of Health.

Ethics

The experimental protocols for mouse studies were approved by the Committee of Animal Care and Use of the National Cancer Institute (ACUC) in Frederick, Maryland, USA (Approval number: 17-072; Approval date: 01/23/2018).

References

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简介

[摘要] 原代培养小鼠海马神经元是一种非常有用的体外模型用于研究神经元的发育,轴突和树突的形态,突触功能,以及许多其他神经元的特征。这里我们描述了从小鼠胚胎海马(E17.5/E18.5)产生初级神经元的一步一步的过程。根据不同的实验目的,用该方法产生的海马神经元可以在不同的组织培养皿中进行培养,并能在不到一周的时间内产生一个可靠的来源。该方案涵盖了神经元培养物的制备、培养和鉴定的所有必要步骤,包括解剖器械的说明、胚胎分离的手术程序、培养条件以及培养物纯度和分化的评估。通过分析培养6天时的钙显像动力学来评估神经元的活性。

[背景] 海马体是一个非常典型的大脑结构,对重要的大脑功能如记忆、空间导航、情绪记忆和学习至关重要。从解剖学上讲,小鼠海马体有一个清晰的C形结构,很容易定位和分离。在细胞水平上,它主要由锥体细胞组成,与其他脑区相比,中间神经元和胶质细胞较少(Kaech和Banker,2006)。因此,海马体是从野生型或基因工程小鼠模型中产生高纯度原代神经元培养物的理想区域,可用于疾病建模或研究神经元功能的多个方面,如突触传递和电生理特性、对神经毒性的敏感性,分化与衰老(;;;;)。Busche,2018Koyama和Ikegaya,2018Molnar,2011Wu等人,2019Rush等人,2020年

已经制定了许多协议,通过与神经胶质喂食器共同培养神经元来产生皮层和海马神经元(Kaech和Banker,2006),描述了用水凝胶微纤维封装的星形胶质细胞的三维神经元培养系统(Kim等人,2020年),长期向培养基中补充生长因子神经元培养(Ray et al.,1993),或从出生后的小鼠中产生神经元(Beaudoin et al.,2012)。根据实验的目的,每种方法都有其优缺点。在这里,我们扩展并详细描述了我们多年来开发并成功应用的海马神经元生成协议(例如,Dorsey等人,2006年;Tomassoni Ardori等人,2019年)。我们主要致力于为胚胎晚期(E17.5-E18.5)获得原代海马神经元提供一种简单、简单、可靠的方法。我们说明了整个准备过程,包括在其他方案中经常被忽略的方面,如解剖器械的描述和胚胎切除和解剖的手术程序。我们报告了在培养6天后通过检测神经元分化标志物和细胞钙成像动力学来分析神经元的分化和功能。此外,我们还显示了神经细胞培养纯度水平与胶质细胞抑制剂阿糖胞苷(Ara-C)治疗前后的关系。

这个循序渐进的方案允许实验者在6天内获得高纯度的分化海马神经元。

关键字:原代培养, 海马神经元, 海马分离, 原代神经元, 小鼠, 神经元钙成像

材料和试剂


 


下表提供了我们实验室中经常使用的材料和设备的示例。类似规格的试剂和设备也可以使用。


1.     聚-D-赖氨酸/层粘连蛋白cellware 12 mm圆形盖玻片(康宁,目录号:354087)


2.     无菌巴斯德玻璃移液管(Thomas Scientific,目录号:1215D99)


3.     24井盘(康宁,目录号:3524)


4.     35mm TC处理培养皿(康宁,目录号:353001)


5.     100 mm TC处理培养皿(康宁,目录号:430167)


6.     SD100-4号一次性玻片


7.     50毫升锥形管(康宁,产品编号:430828)


8.     15ml锥形管(康宁,产品编号:430790)


9.     InvitrolonTM PVDF/滤纸三明治(用于微型凝胶)(赛默飞世尔科学公司,目录号:LC2005)


10.  E17.5/E18.5小鼠胚胎


11.  神经基础培养基(Thermo Fisher Scientific,目录号:21103-049)


12.  B27增补件(赛默飞世尔科技公司,目录号:17504-044)


13.  杜尔贝科改良高糖鹰牌培养基(DMEM)(赛默飞世尔科技公司,目录号:11965-092)


注:此DMEM配方包含4500 mg/L葡萄糖、L-谷氨酰胺和酚红。


14.  蒸馏水(赛默飞世尔科技公司,目录号:15230-162)


15.  聚-D-赖氨酸(Sigma-Aldrich,目录号:P6407)


16.  胎牛血清(非热灭活)(Omega Scientific,目录号:FB-01;或其他品牌FBS)


17.  0.25%胰蛋白酶EDTA(Thermo Fisher Scientific,目录号:25200-056)


18.  青霉素/链霉素(P/S)10000 U/ml(100倍浓缩)(赛默飞世尔科技公司,目录号:15140-122)


19.  杜尔贝科磷酸盐缓冲盐水(DPBS)(赛默飞世尔科技公司,目录号:14190-144)


20.  台盼蓝(Sigma-Aldrich,目录号:93595)


21.  阿糖胞苷(Ara-C)(Sigma-Aldrich,目录号:C6645)


注:储备液在浓度为50mg/ml的水中重新配制,等分并储存在-20°C。


22.  抗微管蛋白-β-III抗体(小鼠单克隆)(Covence,目录号:MMS-435P)


23.  抗GFAP抗体(兔多克隆)(安捷伦(Dako),目录号:Z0334)


24.  阿历克斯抗-21G抗-21G抗-21G抗阿历克斯兔抗-48L二级抗体目录


25.  Alexa FluorTM 647驴抗鼠IgG(H+L)二级抗体(Thermo Fisher Scientific,目录号:A-31571)


26.  DAPI(英维特罗根,目录号:D1306)


27.  多聚甲醛(Sigma-Aldrich,目录号:P6148)


28.  TritonTM-X100(Sigma-Aldrich,目录号:T8532)


29.  驴血清(Sigma-Aldrich,目录号:S30-100ML)


30.  牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)


31.  荧光安装介质(Dako,目录号:S3023)


32.  乙醇溶液70%


33.  鼠层粘连蛋白,天然(Thermo Fisher Scientific,目录号:23017-015)


34.  样品缓冲液,Laemmli 2x浓缩液(Sigma-Aldrich,目录号:S3401)


35.  NuPAGE 4至12%,Bis-Tris,迷你蛋白凝胶(Thermo Fisher Scientific,目录号:NP0321PK2)


36.  吐温20(Sigma-Aldrich,目录号:P1379)


37.  脱脂奶粉(圣克鲁斯生物技术公司,目录号:sc-2324)


38.  Super Signal West Dura超长持续时间基板(Thermo Fisher Scientific,目录号:34076)


39.  抗TrkB抗体(milipore,目录号:07-225)


40.  抗Rbfox1抗体克隆1D10(Millipore,目录号:MABE985)


41.  抗Rbfox3(NeuN)抗体(Millipore,目录号:MAB377)


42.  抗鼠IgG,HRP结合物(Millipore,目录号:AP192P)


43.  抗兔IgG,HRP结合物(Millipore,目录号:AP182P)


44.  1毫米Fluo-4二甲基亚砜溶液(赛默飞世尔科学公司,目录号:F14217)


45.  PowerLoad浓缩液100x(赛默飞世尔科技公司,目录号:P10020)


46.  水溶性丙磺舒(赛默飞世尔科技公司,目录号:P36400)


 


设备


 


1.     解剖罩(NuAire,型号:NU-201-430)


2.     层流罩(NuAire,型号:NU-425-400,II级,A/B3型)


3.     立体显微镜(奥林巴斯,型号:SZH-ILLK)


4.     倒置组织培养显微镜(Nikon,型号:Chipot)


5.     细胞培养二氧化碳培养箱(三洋,型号:MCO-17AIC)


6.     外科剪刀,锋利(精细科学工具,目录号:14002-16)


7.     学生标准型镊子(精细科学工具,目录号:91100-12)


8.     锋利的细剪刀(精细科学工具,目录号:14060-10)


9.     Graefe镊子(精细科学工具,目录号:11052-10)


10.  Dumont#7镊子(精细科学工具,目录号:11272-30)


11.  细胞计数器(Nexcelom Bioscience,Cellometer Vision)


12.  离心机(Thermo Scientific,型号:CL2)


13.  组织培养罩(NuAire,型号:NU-425-400)


14.  共聚焦显微镜系统(蔡司,型号:LSM780)


15.  本生灯(HanauTM Touch-O-Matic)


16.  Branson 1800声波仪(Branson)


17.  XCell SureLock微型细胞蛋白质电泳(Thermo Fisher Scientific,目录号:EI0001)


18.  GeneGnome XRQ化学发光成像系统(Syngene)


 


程序


 


笔记:


a。该方法描述了从E17.5/E18.5小鼠胚胎中产生原代海马神经元所需的关键步骤。每一步之后都包含了实用的提示,以便更好地指导读者完成整个过程。


b。海马神经细胞的最终培养可根据不同的培养基片或玻片进行。


 


A、 用聚-D-赖氨酸涂覆组织培养皿/培养板


1.     将50ml无菌组织培养级水添加到5mgγ射线照射和细胞培养试验的聚-D-赖氨酸瓶(最终工作浓度为0.1mg/ml),在无菌层流罩中重组聚-D-赖氨酸。


2.     在培养皿表面均匀涂上0.1mg/ml聚-D-赖氨酸溶液,室温下在层流罩中培养20分钟。


3.     通过抽吸去除聚-D-赖氨酸溶液,并用无菌组织培养级水彻底冲洗表面两次。


4.     在电镀电池之前,将盘子/盘子置于层流罩下约1小时,使其表面完全干燥。


注:如果玻璃盖玻片上的电镀槽使用商用聚-D-赖氨酸/层粘连蛋白预涂12 mm盖玻片。用无菌层流罩中的无菌镊子将聚-D-赖氨酸/层粘连蛋白涂层的盖玻片直接转移到24孔板的孔中,然后再进行神经元电镀。或者,可以通过将12 mm的常规盖玻片放入24个孔板孔中并在层流罩中用0.1 mg/ml聚-D-赖氨酸覆盖20分钟来涂覆。之后,用PBS清洗两次,并用10µg/ml层粘连蛋白溶液在37°C下覆盖至少4小时,方法是将盖片放在培养箱中,直到细胞准备好进行电镀。在电镀细胞前用PBS冲洗两次,以防止培养表面干燥。将1ml PBS加入1mg层粘连蛋白粉末中,用PBS稀释至10µg/ml层粘连蛋白,制成1 mg/ml层粘连蛋白储备液。在盘子里涂上盖玻片可以避免在盘子之间转移时刮掉涂层。


 


B、 处死孕鼠,取出E17.5/E18.5胚胎


1.     根据动物护理和使用委员会(ACUC)批准的方案对怀孕的老鼠实施安乐死(CO2安乐死或异氟醚麻醉,然后砍头或颈椎脱位)。


2.     把动物固定在一个干净的手术区域,用70%的乙醇清洁腹部。


3.     为了暴露胚胎,使用外科剪刀(图1A)在腹部中部进行切口(图1A),从耻骨区开始,一直到腹腔末端,如图2A中虚线所示。


注:用手术剪进行切口时,用镊子(图1B)夹起皮肤层。这将有助于避免直接位于下面的胚胎受到意外伤害。


 






图1。用于制备海马神经元的解剖仪器。A、 方案步骤B3-B6中使用的外科剪刀。B、 方案步骤B3-B6和C1中使用的标准型镊子。C、 步骤C1中使用的细剪刀。D、 在步骤C2-C3中使用的弯曲镊子(抓钳)。E、 步骤C4和C5中使用的Dumont镊子。


 






图2。E17.5/18.5妊娠动物胚胎手术切除的制备。A、 用白色虚线显示切口位置的固定动物(步骤B3);B.腹部切口后子宫暴露(步骤B3)。


 


4.     用镊子和手术剪刀(图1A-1B)小心地暴露含有胚胎的整个子宫(子宫角)(图2B),并立即将子宫转移到装有冰PBS的100 mm培养皿中(图3A)。


注:用镊子抓住子宫并将其拉出腹部,另一只手用外科剪刀切断任何粘连和宫颈连接(图2B箭头所示)。


5.     继续用镊子和手术剪刀取出单个胚胎,小心地取出胎盘和卵黄袋。将胚胎移入一个新的100毫米的装有冰PBS的培养皿中,以清洗胚胎并除去血液和胚胎液(图3B)。


6.     用镊子(图1B)固定胚胎,用外科剪刀切开头部(图3C)。将头部转移到一个新的100毫米培养皿中,其中含有10%胎牛血清和1%抗生素(P/S)。


注意:从这一步开始,重要的是不断地将生物材料转移到新的含有抗生素的培养基中,以降低污染的可能性。在本方案所示的图中,有意使用PBS代替含有酚红的介质,仅为了获得更清晰的图像(图3A、图4、图5和图6)。


 






图3。从子宫中分离胚胎。A、 分离的胚胎(B5。B步);分离的胚胎(B5。B步);分离的胚胎。D、 胚胎头部的侧视图。白色虚线表示从头骨后部(黑色剪刀)开始执行步骤C1中所述的横向切割,同时用镊子将胚胎头部固定在鼻子区域(黑色箭头)。


 


C、 海马解剖


1.     移到无菌层流气流的解剖柜中,准备一个100毫米的含有10%胎牛血清和1%抗生素(P/S)的冷冻完整DMEM的培养皿,用于大脑解剖。每次将一个胎头移入含有完整DMEM培养基的100 mm培养皿中,轻轻地从胎头中取出大脑(图4A)。具体来说,用一只手用镊子(图1B)捏住头部的口/鼻区域(图3D中的黑色箭头)。另一只手用细剪刀(图1C)从颅骨后部进入(图3D中为黑色剪刀)并进行横向切割(沿图3D中白色虚线所示嘴/鼻水平的横切面),以释放基底脑区。将胎头倒置,露出大脑的游离基底区。继续用两把镊子(图1B)轻轻地拉开颅骨,小心地取出整个大脑(图4A)。将大脑转移并收集到一个新的100毫米培养皿中,盛有干净的、冰镇的完整DMEM培养基。每次脑切除后更换培养基,排除血液。


2.     一次将一个大脑转移到一个35毫米的盛满冰镇DMEM培养基的皿盖中,然后在解剖立体镜下移动。


注:与35mm培养皿底部相比,使用解剖仪器时,35 mm培养皿盖的较低深度允许更好的移动。


3.     使用两个弯曲镊子(图1D):用一个镊子(图4A中的白点)将大脑固定在一侧,并沿中线(图4A中的虚线)插入第二个镊子,以分离大脑的两个半球。继续使用弯曲镊子轻轻地分开和翻转大脑的两个半部分,以便暴露和显示海马区(图4B-4C)。


注意:由于胚胎组织的脆弱性和柔软性,在试图解剖海马区时,大脑很容易受到损伤:在处理和握住胚胎脑时要小心。在立体镜上进行几次练习将有助于快速提高解剖技能。


 






图4。海马体的定位。A、 解剖胚胎脑。白色虚线表示用一个弯曲镊子将两个大脑半球分开,同时用第二个弯曲镊子(白点)将大脑保持在一侧(步骤C3)。B、 分离的大脑半球。白色箭头表示每个半球海马区的位置(步骤C3)。C、 高倍图像显示一个大脑半球的海马区,用白色虚线表示。


 


4.     小心地从每个大脑半球移除脑膜(图5)。


笔记:


a。使用两个Dumont镊子(图1E)。用一把镊子夹住整个大脑,用第二把镊子轻轻地剥离脑膜。从捏嗅球区开始,逐渐向后拉,以便完全移除脑膜(图5)。在做这个手术的时候,轻轻地握住大脑,根据脑膜脱落的容易程度,可能有必要多次改变控制点。


b。正确地去除脑膜是很重要的,以消除尽可能多的非神经元细胞对神经元培养的贡献。


 






图5。脑膜切除术。箭头显示海马和皮质区域以及嗅球和脑膜从一个大脑半球移除(步骤C4)。


 


5.     使用Dumont镊子沿着海马体边缘进行精细切割,以完全释放海马体区域(图5-6)。用弯曲的镊子轻轻地将每个海马移到一个新的35毫米的组织培养皿中,培养皿中含有2ml的冰镇无血清DMEM培养基和1%的抗生素(P/S),并保存在冰上。


注:海马体可通过其典型的C形解剖结构和立体镜下与海马体相连的皮质区的光对比度差异来区分(图5-6)。在移除海马体之后,如果需要的话,可以从相同的胚胎中收集皮质来准备皮质神经元(本方案中没有描述)。


 


D、 海马胰蛋白酶化


1.     用两个Dumont镊子将海马组织切成小块(理想情况下,每个海马体约2-3 mm3),在含有2毫升冰镇无血清DMEM培养基的35毫米组织培养皿中切碎海马组织(图6C)。


注:重要的是收集并保存在无血清DMEM培养基中(步骤C5和D1),以避免在下一步(步骤D2)中抑制胰蛋白酶EDTA活性。


 






图6。分离海马体(步骤C5和D1)。A、 从一个胚胎脑中分离出海马。B、 海马区高倍图像显示典型的C形解剖结构。C、 碎海马。用于胰蛋白酶消化的海马组织块(步骤D1)。


 


2.     移到带有无菌层流气流的组织培养罩中,用移液管将含有碎海马的培养基转移到50 ml的无菌锥形管中。轻轻地将2ml 0.25%胰蛋白酶EDTA加入海马,并将试管置于37°C的水浴中25分钟(最终胰蛋白酶浓度为0.125%)。


注:在胰蛋白酶消化25分钟的过程中,切碎的海马体在重力作用下自然沉积到50毫升锥形管的底部。


3.     准备一个15毫升的无菌锥形管,其中含有2毫升温热(37摄氏度)DMEM,含10%胎牛血清和1%抗生素(P/S)。消化结束后,小心地将50毫升消化管移回组织培养罩中,不干扰或重新消耗海马沉积物,并使用1毫升针尖的吸管,仅将海马沉积物转移到含有2毫升完全培养基的新的15毫升锥形管中。


注:用1毫升针头直接接触50毫升试管底部的海马沉积物。轻轻吸气,只转移管底部的海马体,不含消化液。新的2ml培养基中所含的胎牛血清将使胰蛋白酶失活并停止其酶活性。


 


E、 海马组织的磨碎


1.     用本生灯点燃抛光无菌玻璃巴斯德吸管,并产生一个较小的开口和光滑的尖端,以分离海马组织(图7)。使用如图7B所示的火抛光吸管慢慢地、轻轻地研磨海马体。用吸管上下吸6到8次。


注:培养海马是获得良好细胞产量和细胞活力的关键步骤。火抛光后,一定要让玻璃吸管冷却下来再磨碎海马体。海马体块应该在上下吸6到8次后完全破碎。


2.     再次使用本生灯,准备一个新的火拔玻璃巴斯德吸液管,其尖端较长且呈锥形(图7C)。使用该移液管缓慢地向上和向下吸移海马细胞浆,以释放单个细胞。获得单细胞悬浮液后,立即进行程序F。将单细胞悬浮液保存在组织培养罩中,室温下无菌层流气流,同时计数细胞。


注:使用带有较长和锥形尖端的火拉巴斯德吸液管(图7C)会损坏细胞并影响细胞活力。


 






图7。玻璃巴斯德吸管头(步骤E1和E2)。A、 完整的玻璃巴斯德吸管头。B、 火焰抛光玻璃巴斯德吸液管,尖端光滑且较小。C、 火抛光玻璃巴斯德吸管锥形和超小头。


 


F、 细胞计数、细胞播种和细胞培养


1.     收集50μl细胞悬浮液,并添加5μl台盼蓝染料以评估细胞活性。使用单元格计数器计数单元格。


注:细胞存活率在活细胞的70-90%之间。例如,从8个胚胎中提取的神经元制剂的细胞活力和细胞计数是总共5 x 106个细胞中约4 x 106个活细胞(细胞活力=80%)。


2.     将剩余的海马细胞悬浮液在350 x g下离心5分钟。丢弃上清液,并通过在含B27补充剂的温热神经基础培养基中轻轻上下吸管重新悬浮细胞颗粒。根据将要使用的组织培养板调整培养基的体积。使用大约260000个细胞/平方厘米的播种密度。将细胞接种在先前涂有聚-D-赖氨酸的组织培养皿/平板中。


注意:细胞产量约为400000-600000个细胞/胚胎。在500 ml神经基础培养基中加入10 ml B27增补剂和5 ml P/S抗生素(100倍浓缩液),制备完整的神经元培养基。一些协议(例如。, Ruhl等人,2019年)建议在含10%FBS的DMEM培养基中预镀1小时,以促进神经元与板的粘附。然而,我们已经发现直接在神经基础培养基中加入B27补充剂就足以使神经元粘附良好。


3.     在初镀18~24小时后,加入阿糖胞苷(Ara-C),停止分裂细胞(非神经元细胞)的增殖。去除培养基(含有B27补充剂的神经基础培养基)的一半体积,并用含有2µM Ara-C(1µM Ara-C最终浓度)的新鲜培养基替换。


注:未将Ara-C添加到原代神经元培养物中会严重影响培养物的纯度(图8B和8F)。在神经元培养液中加入Ara-C可观察到一些有丝分裂细胞死亡。


4.     培养原代海马神经元,每48~72h更换一半培养基,初次治疗后更换培养基时不必继续添加Ara-C。培养6天后,原代神经元将发育良好并分化为两种形态,尤其是典型神经元标记物的表达,如微管蛋白-β-Ⅲ、NeuN、Rbfox1、神经营养素受体和其他一些神经元蛋白质(Tomassoni Ardori et al.,2019)(图8和图9)。


笔记:


a。只需更换一半的培养基,就可以保持生长因子在神经元条件培养基中自然分泌和存在。


b。虽然在原代细胞培养物制备过程中不可能绝对防止微生物污染(细菌、支原体、真菌),但这些步骤可以大大提高结果:i)在无菌环境中工作;ii)使用适当的无菌技术,包括戴无菌手套、使用无菌玻璃器皿和一次性移液管;以及小贴士;iii)保持所有设备清洁(解剖显微镜、培养箱、离心机和水浴)。


 


 


图8。培养中神经元的发育和分化。A、 原代海马神经元体外培养1~6天的Brightfield图像。注意在体外(放大20倍)神经元网络的复杂性随着时间的推移而增加。B、 体外培养6天后海马神经元的Western印迹分析。免疫印迹采用针对分化神经元标志物的抗体:BDNF受体TrkB胞外区,检测所有TrkB亚型,Rbfox1,βIII微管蛋白(b-III-Tub)和Rbfox3(NeuN)。分子量标记显示在左边。


 


G、 免疫印迹分析检测分化神经元的标记物(图8B)


1.     体外培养6天后,从含有神经元的24孔板的一个孔中取出培养基。


2.     用PBS冲洗细胞一次。


3.     通过直接向孔中添加150µl 2x Laemmli样品缓冲液来溶解细胞。


4.     将细胞裂解液转移至1.5 ml试管中,超声处理10分钟,将基因组DNA切碎并消除粘度。


5.     将样品在95°C下加热5分钟,以促进蛋白质变性。


6.     将10µl样品每通道装入4-12%NuPAGE预铸凝胶中,并在120 V下运行凝胶。


7.     将凝胶转移到PVDF膜上。


8.     在室温下用5%脱脂牛奶在TBS吐温(0.1%)中封闭薄膜1小时。


9.     在4°C的温度下,轻轻摇动,用特定的初级抗体孵育一整夜。在TBS吐温(0.1%)缓冲液中,在5%脱脂牛奶中稀释1:1000。


10.  用TBS Tween(0.1%)缓冲液清洗薄膜三次,5分钟。


11.  用HRP结合的次级抗体在室温下孵育1h,同时轻轻摇动。在TBS吐温(0.1%)缓冲液中稀释1:5000的二级抗体。


12.  用TBS Tween(0.1%)缓冲液清洗薄膜三次,5分钟。


13.  用增强化学发光(ECL)基质培养膜,用化学发光成像系统或胶片曝光获得图像。


 


H、 免疫荧光法对海马神经元的表型分析(图9)


1.     培养6天后,将培养基从含有多聚-D-赖氨酸/层粘连蛋白预涂层的12 mm带海马神经元的盖玻片的24孔板中取出。用PBS冲洗电池,并用4%多聚甲醛/PBS溶液在4°C下固定30分钟。


2.     用PBS无钙/镁(PBS-/-)冲洗细胞3次,持续5分钟。


3.     在室温下使用封闭溶液1小时。


注:用10%正常驴血清(v/v)、0.1%TritonTM-X100(v/v)和0.1%BSA(w/v)制备封闭溶液。


4.     在4°C下用特异性初级抗体培养细胞过夜。


注:在含有1%正常驴血清、0.1%TritonTM-X100(v/v)和0.1%BSA(w/v)的PBS-/-溶液中稀释初级抗体。抗GFAP抗体的稀释度为1:1000,抗微管蛋白β-III抗体的稀释度为1:200(见材料和试剂)。


5.     用PBS-/-冲洗细胞3次,持续5分钟。


6.     在室温下用特异性二级抗体和DAPI染色(0.5µg/ml)孵育2小时。Alexa Fluor 488驴抗兔IgG(H+L):抗GFAP抗体的次级抗体;Alexa Fluor 647驴抗小鼠IgG(H+L):抗微管蛋白-β-III抗体的次级抗体。使用与步骤G4相同的溶液稀释次级抗体1:250。


7.     用PBS-/-冲洗细胞3次,持续5分钟。


8.     倒置盖玻片,并使用Dako荧光安装介质将其安装到玻璃载玻片上。当安装介质干燥后,样品可以通过共焦成像进行分析。


注:尽管在本方案中,我们表明,在培养6天内可获得分化的海马神经元,我们建议使用阿糖胞苷提高神经元培养纯度(图8和图9),但在某些情况下,与胶质细胞共培养神经元可进一步支持神经元成熟和长期存活(Kaech和Banker,2006年Kim等人,2020年;).






图9。原代海马神经元培养6天后的免疫荧光染色(G程序)。用(A-D)或不加(E-H)阿糖胞苷(Ara-c1µM)培养神经元,并对胶质细胞标记物胶质纤维酸性蛋白(GFAP为绿色)、神经元标记物βIII微管蛋白(红色为b-III-TUB)和细胞核染色剂(DAPI为蓝色)染色。注意6天后未经Ara-C处理(F)的神经元培养中,GFAP阳性细胞数量显著增加。用1µM Ara-C培养的神经元的高倍放大图像(I-K),并对βIII微管蛋白(b-III-TUB为绿色)和细胞核染料(蓝色为DAPI)染色。体外培养6天后观察神经元网络的复杂性。


 


一、 海马神经元钙显像功能表型特征(图10和视频1)


功能分化神经元的进一步测试包括自发或谷氨酸诱导去极化后体外发生的钙动态成像(Grienberger和Konnerth,2012)。


1.     Fluo-4负载缓冲液的制备:为制备1 ml负载缓冲液,在10µl PowerLoad(100x浓缩液)中加入1µl的Fluo-4储备液(Fluo-4储备液:1 mM在二甲基亚砜中;最终浓度为1µm)。然后添加10µl丙磺舒(储备溶液250 mM;最终浓度2.5 mM)和979µl ECS缓冲液(140 mM NaCl、4.7 mM KCl、2 mM NaHCO3、1 mM NaH2PO4、1.2 mM MgCl2、1.5 mM CaCl2、3 mM葡萄糖和10 mM HEPES,pH 7.4)。


2.     体外培养6天后,用含有海马神经元的盖玻片从24孔板中取出培养基。用250µl的Fluo-4负载缓冲液在37°C下培养每个盖玻片30分钟,然后在37°C的ECS缓冲液中洗涤30分钟,以便使Fluo-4脱酯化和活化。


3.     将记录室中的流量调节至0.5 ml x min,并将温度设置为37°C。


4.     将盖玻片放入录音室并选择合适的区域。


5.     设置焦距,将激光强度(480 nm)设置为较低的可能值和积分时间(像素停留时间),以便在以2个图像x秒的共焦模式开始时间序列采集之前,采集时间小于500毫秒。


6.     记录神经元的自发活动,然后在1毫米处施加谷氨酸盐,持续10秒,去极化并观察场中的所有细胞。


7.     要进行图像分析,请在ImageJ(或斐济)中导入文件https://imagej.net/Fiji/Downloads);将查找表设置为彩虹RGB;调整图像亮度和对比度;打开ROI管理器(分析/工具/ROI管理器);选择一个合适的区域形状覆盖感兴趣的神经元,然后选择Images/Stacks/Plot Z-axis Profile来绘制所选ROI随时间变化的光子密度。将ROI添加到ROI管理器,并继续执行另一个ROI。密度图可以导出到电子表格,以便进一步量化。


 






图10。体外培养6天后原代海马神经元的钙显像(程序一)。 用钙指示剂Fluo-4(A)测定原代海马神经元钙荧光强度随时间的变化。在用1 mM谷氨酸盐(蓝色条)治疗前,注意神经元的自发活动。(A)中分析的单个神经元用(B)中的数字表示。在(C)或(D)用1 mM谷氨酸盐刺激前(D)后神经元培养中钙荧光的典型图像。






视频1。体外培养6天后原代海马神经元的钙离子实时成像(图10;程序I)


 


致谢


 


我们感谢艾琳·索森和安蒂·凯洛希·佐藤康钦对手稿的批判性阅读。这项工作得到了国家癌症研究所癌症研究中心NIH校内研究计划的支持。


 


相互竞争的利益


 


作者声明没有竞争性的利益,提到特定的材料、试剂和设备并不意味着得到美国国立卫生研究院的认可。


 


伦理学


 


小鼠研究的实验方案经美国马里兰州弗雷德里克国家癌症研究所(ACUC)动物护理和使用委员会批准(批准号:17-072;批准日期:2018年1月23日)。


 


工具书类


 


1.     Beaudoin,G.M.,3rd,Lee,S.H.,Singh,D.,Yuan,Y.,Ng,Y.G.,ReichardL.F.和Arikkath,J.(2012年)。从出生后早期小鼠海马和皮层培养锥体神经元。Nat协议7(9):1741-1754。


2.     Busche,M.A.(2018年)。阿尔茨海默病小鼠海马神经元双光子成像研究。方法分子生物学1750:341-351。


3.     Dorsey,S.G.,Renn,C.L.,Carim Todd,L.,Barrick,C.A.,Bambrick,L.,Krueger,B.K.,Ward,C.W.和Tessarolo,L.(2006年)。体内恢复TrkB.T1受体截短的生理水平拯救三体小鼠模型中的神经元细胞死亡。神经元51(1):21-28。


4.     Grienberger,C.和Konnerth,A.(2012年)。神经元钙成像。神经元73(5):862-885。


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Copyright Tomassoni-Ardori et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tomassoni-Ardori, F., Hong, Z., Fulgenzi, G. and Tessarollo, L. (2020). Generation of Functional Mouse Hippocampal Neurons. Bio-protocol 10(15): e3702. DOI: 10.21769/BioProtoc.3702.
  2. Tomassoni-Ardori, F., Fulgenzi, G., Becker, J., Barrick, C., Palko, M. E., Kuhn, S., Koparde, V., Cam, M., Yanpallewar, S., Oberdoerffer, S. and Tessarollo, L. (2019). Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels. Elife 8: e48673.
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Victoria Hewitt
Columbia University
Thanks for sharing this protocol. Have you ever done any staining to confirm you do not end up with any cortical neurons in your prep? If so could you share which markers you used?
2021/6/14 8:21:54 回复
Francesco Tomassoni-Ardori
Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick

Hi Victoria! The straight answer to you question is no, we have never tried any specific cortical marker on those cultures. The hippocampus has a very well defined structure which allows a clean dissection (as you can see in Fig.6). Being the "starting material" so pure, you would expect a minimal or negligible cortical contamination, if any. All the best!

2021/6/15 6:57:25 回复


Victoria Hewitt
Columbia University

Thanks for getting back to me - I'm trying to pair this with an electroporation before plating at E15.5 so the dissection is a little more fiddly but they grow a lot more robustly than the cortical neurons. It would just be nice to have a way to be sure.
Thanks
Victoria

2021/6/16 9:32:48 回复