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An ex vivo Model of Paired Cultured Hippocampal Neurons for Bi-directionally Studying Synaptic Transmission and Plasticity

成对培养的海马神经元的离体模型，用于双向研究突触传递和可塑性

RS Ruslan Stanika
GO Gerald J. Obermair

发布: 2023年07月20日第13卷第14期 DOI: 10.21769/BioProtoc.4761 浏览次数: 2398

评审: Verena BurtscherMohammed Mostafizur RahmanWilly R Carrasquel-Ursulaez

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Abstract

Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons.


Key features

• Preparation of ex vivo paired cultured hippocampal neurons.

• Bi-directional electrophysiological recordings of synaptic transmission and plasticity.

• Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel α2δ-2 subunit).


Graphical overview


Keywords: Primary hippocampal culture (原代海马培养) search Paired patch clamp recording (成对膜片钳记录) search Synaptic transmission (突触传递) search Calcium channel (钙通道) search α2δ subunit (α2δ亚基) search Viral infection (病毒感染) search Electrophysiology (电生理学) search Short-term plasticity (短期可塑性) search Postsynaptic currents (突触后电流) search Paired pulse facilitation and depression (成对脉冲促进和抑制) search

Background

Signal transmission between neurons occurs via neurotransmitter release into the synaptic cleft. The temporal and spatial relation of pre- and post-synaptic firing modulates the strength of synaptic connections between neurons (Deperrois and Graupner, 2020). Two types of synaptic activity can be registered at the single cell level. First, miniature excitatory or inhibitory postsynaptic currents (mEPSC or mIPSC, respectively) appear as result of spontaneous local fusion of single synaptic vesicles. Second, excitatory and inhibitory postsynaptic currents (EPSC or IPSC, respectively) can be recorded in response to action potential firing by presynaptic glutamatergic or GABAergic neurons, respectively. Analysis of miniature postsynaptic potentials provides information on the amount and density of synaptic connections (frequency), as well as the postsynaptic receptor abundance (amplitude), and hence helps to study elementary synapse properties. However, higher levels of synaptic function, including the responses of synapses in regard to action potential firing, as well as short- and long-term adaptations of synaptic strength, require the analysis of evoked synaptic transmission. For example, paired-pulse stimulation protocols can serve as a basic model for studying short-term synaptic plasticity ex vivo (Bouteiller et al., 2010). Evoked synaptic transmission and plasticity in specific neuronal pathways can typically be studied in brain slices (Wang and Baudry, 2019). Alternatively, synaptic transmission and plasticity can be studied in cultured neurons, such as by employing optogenetic activation of neuronal cell populations (Barral and Reyes, 2017). In an acute brain slice, which is the standard model for the electrophysiological analysis of synaptic functions, presynaptic stimulation and postsynaptic responses can only be analyzed in one direction. However, as synaptic plasticity involves the possibility of changes in both pre- and post-synaptic components, one-directional measurements limit the study of mechanisms involved in modulating plasticity.

Here, we describe a protocol for culturing simple networks of paired hippocampal neurons for the bi-directional electrophysiological analysis of synaptic functions. This cellular ex vivo model has the following advantages: first, it allows the easy identification of the innervated cells. In classical neuronal cell cultures employing dispersed neurons, this is inherently difficult due to excessive branching of the axons and the possibility of hetero-synaptic innervation of the target neuron. Second, due to the defined simple network, all synapses are formed between the paired neurons. This results in increased amplitudes of postsynaptic responses and hence allows the reliable detection of changes in postsynaptic receptor function. Third, both cells of the cultured paired network can function as presynaptic (stimulated) and postsynaptic (innervated) neurons. Hence, this method allows recording synaptic transmission bi-directionally. This is particularly relevant in the context of genetic manipulations of one of the two paired neurons: as one of the paired neurons can be genetically modified by overexpression or knockdown of specific proteins, bi-directional stimulation protocols allow analyzing pre- and post-synaptic consequences in comparison with wild-type synaptic connections in the same neuronal network. As a proof of principle, we altered the expression of α2δ proteins, which act, on the one hand, as auxiliary subunits of voltage-gated calcium channels (Geisler et al., 2015; Ablinger et al., 2020; Dolphin and Obermair, 2022), and on the other hand as critical synaptic organizers (Eroglu et al., 2009; Geisler et al., 2019; Schöpf et al., 2021; Ablinger et al., 2022). Hence, we employed cultured paired hippocampal neurons to investigate the role of a splice variant of the α2δ-2 isoform in the trans-synaptic regulation of synapse formation and synaptic transmission, including short-term synaptic plasticity.

Materials and reagents

Animals

Timed pregnant wild-type mice (BALB/c, gestational age 16–17 days; Charles River Laboratories, Sulzfeld, Germany).


Biological materials

Lentiviral particles, carrying RNA encoding for the α2δ-2_ΔE23 splice variant and soluble eGFP as fluorescent marker (Geisler et al., 2019). Lentiviral particles were generated as previously described (Nasri et al., 2014; Benskey and Manfredsson, 2016).

Critical: Lentiviruses are classified as a biosafety level 2 (BSL-2) organism.


Materials

  1. Surgical scissors, sharp blunt, straight 14.5 cm (Fine Science Tools, catalog number: 14001-14)

  2. Tissue forceps, slim 1 × 2 teeth 10 cm (Fine Science Tools, catalog number: 11023-10)

  3. Fine scissors, sharp, curved 10.5 cm (Fine Science Tools, catalog number: 14061-10)

  4. Fine scissors, sharp, straight 10.5 cm (Fine Science Tools, catalog number: 14060-10)

  5. Dumont #5 standard forceps (Fine Science Tools, catalog number: 11251-30)

  6. Dumont #5 biology forceps (Fine Science Tools, catalog number: 11252-30)

  7. Vannas-Tübingen spring scissors (Fine Science Tools, catalog number: 15004-08)

  8. 18 mm glass coverslips (Marienfeld Superior, catalog number: 0111580)

  9. Rack for coverslips (custom build, Institute of Physiology, Medical University Innsbruck, Austria)

  10. PTFE dish (Carl Roth, catalog number: K837.1)

  11. 12.5 cm filter paper (Carl Roth, catalog number: AP86.1)

  12. Hemacytometer (Neubauer, catalog number: Brand 717805)

  13. 72 μm nylon mesh (Falcon, catalog number: 352350)

  14. T75 flask (Falcon, catalog number: 353810)

  15. Transfer pipette 3.5 mL (Sarstedt, catalog number: 86.1171.001)

  16. 15 mL centrifuge tube (Falcon, catalog number: 352070)

  17. 50 mL centrifuge tube (Falcon, catalog number: 352096)

  18. 60 mm plastic Petri dish (Falcon, catalog number: 353802)

  19. 60 mm Primaria plastic Petri dish (Falcon, catalog number: 353004)

  20. 15 cm glass Petri dish (Duran, catalog number: 237555201)

  21. Pasteur pipette (Assistent, catalog number: 40567002)

  22. 5 mL serological pipette (Sarstedt, catalog number: 86.1253.001)

  23. 10 mL serological pipette (Sarstedt, catalog number: 86.1254.001)

  24. 25 mL serological pipette (Sarstedt, catalog number: 86.1685.001)

  25. 1.5 mL miniature spray (Rene Lezard)

  26. Borosilicate glass with filament (Sutter Instrument, model: BF150-75-10)

  27. 2.5% trypsin (10×) (Gibco, catalog number: 15090-046)

  28. 0.5% Trypsin-EDTA (10×) (Gibco, catalog number: 15400-054)

  29. B-27 supplement (50×) (Gibco, catalog number: 17504-044)

  30. GlutaMAX (Gibco, catalog number: 35050-038)

  31. Horse serum (Gibco, catalog number: 16050-122)

  32. PenStrep (Penicillin-Streptomycin) (Gibco, catalog number: 15140-122)

  33. MEM (Gibco, catalog number: 41090-028)

  34. Neurobasal medium (Gibco, catalog number: 21103-049)

  35. HBSS (10×) (Gibco, catalog number: 14180-046)

  36. HEPES 1 M solution (Gibco, catalog number: 15630-056)

  37. Poly-L-lysine (Sigma, catalog number: P2636)

  38. Ara-C (Sigma, catalog number: C6645)

  39. DNase (Sigma, catalog number: DN-25)

  40. Sodium pyruvate (Sigma, catalog number: P2256)

  41. Paraffin (Carl Roth, catalog number: X880.1)

  42. Gelatine (Fluka, catalog number: 48722)

  43. Nitric acid (Carl Roth, catalog number: 4989.2)

  44. Glucose (Carl Roth, catalog number: HN06.3)

  45. Boric acid (Sigma, catalog number: B6768)

  46. Borax (sodium tetraborate decahydrate) (Sigma, catalog number: B9876)

  47. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.1)

  48. Potassium chloride (KCl) (Carl Roth, catalog number: 6781.3)

  49. Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239.2)

  50. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma, catalog number: M0250)

  51. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.3)

  52. Potassium hydroxide (KOH) (Carl Roth, catalog number: 6751.1)

  53. Gluconic acid, potassium salt (K-gluconate) (Carl Roth, catalog number: 4621.1)

  54. HEPES (Carl Roth, catalog number: 6763.1)

  55. EGTA (Sigma, catalog number: E3889)

  56. ATP, magnesium salt (Sigma, catalog number: A9187)

  57. GTP, sodium salt (Sigma, catalog number: G8877)


Solutions

  1. Pyruvate solution, 100 mM, 50 mL (see Recipes)

  2. 1% gelatine solution, 50 mL (see Recipes)

  3. HBSS, 500 mL (see Recipes)

  4. Glia medium, 500 mL (see Recipes)

  5. Neuronal maintenance medium, 200 mL (see Recipes)

  6. Neuronal plating medium, 200 mL (see Recipes)

  7. 1% DNase solution, 100 mL (see Recipes)

  8. Sodium borate buffer, 500 mL (see Recipes)

  9. Poly-L-lysine solution, 1 mg/mL (see Recipes)

  10. EGTA solution, 0.5 M, 1 mL (see Recipes)

  11. Extracellular solution, 100 mL, adjust pH 7.4 with NaOH (see Recipes)

  12. Intracellular solution, 20 mL, adjust to pH 7.2 with KOH (see Recipes)

  13. Sodium hydroxide solution, 1 M, 5 mL (see Recipes)

  14. Potassium hydroxide solution, 1 M, 5 mL (see Recipes)

  15. 20% glucose solution, 50 mL (see Recipes)

Recipes

  1. Pyruvate solution, 100 mM, 50 mL

    ReagentAmount
    Sodium pyruvate550 mg
    Milli-Q waterAdd to the total volume of 50 mL


  2. 1% gelatine solution, 50 mL

    ReagentAmount
    Gelatine500 mg
    Milli-Q waterAdd to the total volume of 50 mL


  3. HBSS, 500 mL

    ReagentAmount
    HBSS (10×)50 mL
    PenStrep5 mL
    HEPES, 1 M5 mL
    Milli-Q water440 mL


  4. Glia medium, 500 mL

    ReagentAmount
    MEM430 mL
    PenStrep5 mL
    Glucose, 20%15 mL
    Horse serum50 mL


  5. Neuronal maintenance medium, 200 mL

    ReagentAmount
    Neurobasal medium194 mL
    B-27 supplement, (50×)4 mL
    GlutaMAX2 mL


  6. Neuronal plating medium, 200 mL

    ReagentAmount
    MEM172 mL
    Pyruvate solution, 100 mM2 mL
    Glucose, 20%6 mL
    Horse serum20 mL


  7. 1% DNase solution, 100 mL

    ReagentAmount
    DNase1 g
    HBSS100 mL


  8. Sodium borate buffer, 500 mL

    ReagentFinal concentrationAmount
    Boric acid50 mM1.54 g
    Borax (sodium tetraborate)12.5 mM2.376 g
    Milli-Q waterAdd to the total volume of 500 mL


  9. Poly-L-lysine solution, 1 mg/mL

    ReagentAmount
    Poly-L-lysine100 mg
    Sodium borate buffer100 mL


  10. EGTA solution, 0.5 M, 1 mL

    ReagentAmount
    EGTA190.2 mg
    1 M KOHAdd to the total volume of 1 mL


  11. Extracellular solution, 100 mL, adjust pH 7.4 with 1 M NaOH solution

    ReagentFinal concentrationAmount
    NaCl137 mM800.62 mg
    KCl3 mM22.36 mg
    Glucose10 mM180.2 mg
    HEPES10 mM238.3 mg
    CaCl2·2H2O1.8 mM26.46 mg
    MgCl2·6H2O2 mM40.66 mg


  12. Intracellular solution, 20 mL, adjust to pH 7.2 with 1 M KOH solution

    ReagentFinal concentrationAmount
    K-gluconate125 mM585.62 mg
    KCl10 mM14.9 mg
    HEPES10 mM47.66 mg
    MgCl2·6H2O1 mM4.06 mg
    EGTA solution, 0.5M2 mM80 μL
    ATP (Mg)4 mM40.57 mg
    GTP (Na)0.3 mM3.14 mg


  13. Sodium hydroxide solution, 1 M, 5 mL

    ReagentAmount
    NaOH200 mg
    Milli-Q waterAdd to the total volume of 5 mL


  14. Potassium hydroxide solution, 1 M, 5 mL

    ReagentAmount
    KOH280.6 mg
    Milli-Q waterAdd to the total volume of 5 mL


  15. 20% glucose solution, 50 mL

    ReagentAmount
    Glucose10 g
    Milli-Q waterAdd to the total volume of 50 mL

Equipment

  1. Stereo dissection microscope (Olympus, model: SZX2-ILLTQ)

  2. Class II biological safety cabinet (Thermo Scientific, model: HERAsafe KS12)

  3. Suction system (Welch, model: 112037-08)

  4. Safety Bunsen burner (INTEGRA Biosciences, model: Fireboy Plus 144000)

  5. Pipette controller (INTEGRA Biosciences, model: PIPETTEBOY acu 2)

  6. Magnetic stirrer with heating (Phoenix Instrument, model: RMS-10HS)

  7. CO2 incubator (Thermo Scientific, model: HERAcell 240i)

  8. Water baths (VWR, catalog number: 462-0558)

  9. pH meter (inoLab, model: pH 7110)

  10. Micropipette puller (Sutter Instrument, model: P-97)

  11. Microforge (Narishige, model: MF-830)

  12. Ultrapure water purification system (Merck Millipore, model: Milli-Q® EQ 7000)

  13. Universal oven (Memmert, model: UF260)


Experimental setup

  1. CleanBench lab table (TMC, model: TM-63-9012S)

  2. Inverted fluorescent microscope (Olympus, model: IX-83, equipped with LUCPLFLN40XPH/0.6 objective, Lumencor LED lamp, eGFP filter)

  3. Microscope dependent platforms (Sutter Instrument, model: MDM-83-2)

  4. Micromanipulators (Sutter Instrument, model: MPC-325-2)

  5. Two-channel patch clamp amplifier (HEKA Elektronik, model: EPC 10 USB Double)

  6. Quick change chamber RC-41LP (Harvard Apparatus, catalog number: 64-0368)

Software

  1. PatchMaster v2x90.5 (HEKA Elektronik)

  2. FitMaster v2x90.5 (HEKA Elektronik)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Stanika, R. and Obermair, G. J. (2023). An ex vivo Model of Paired Cultured Hippocampal Neurons for Bi-directionally Studying Synaptic Transmission and Plasticity. Bio-protocol 13(14): e4761. DOI: 10.21769/BioProtoc.4761.
  2. Geisler, S., Schöpf, C. L., Stanika, R., Kalb, M., Campiglio, M., Repetto, D., Traxler, L., Missler, M. and Obermair, G. J. (2019). Presynaptic α2δ-2 Calcium Channel Subunits Regulate Postsynaptic GABA(A) Receptor Abundance and Axonal Wiring. J. Neurosci. 39(14): 2581-2605.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 细胞机理 > 突触生理学

生物物理学 > 电生理 > 膜片钳技术

细胞生物学 > 细胞信号传导 > 突触传递

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