Assembly of Synapses: Biomimetic Assays to Control Neurexin/Neuroligin Interactions at the Neuronal Surface

Magali Mondin1, Béatrice Tessier2, Olivier Thoumine2

1 INSERM, Bordeaux Imaging Center, Bordeaux, 2 Université Bordeaux, Interdisciplinary Institute for Neuroscience, Bordeaux
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.19
DOI:  10.1002/0471142301.ns0219s64
Online Posting Date:  July, 2013
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Abstract

The role of adhesion molecules in the assembly of synapses in the nervous system is an important issue. To characterize the role of neurexin/neuroligin adhesion complexes in synapse differentiation, various imaging assays can be performed in primary hippocampal cultures. First, to temporally control contact formation, biomimetic assays can be performed using microspheres coated with purified neurexin or with antibody clusters that aggregate neurexin. These models are combined with live fluorescence imaging to study the dynamics of accumulation of post‐synaptic components, including scaffolding molecules and glutamate receptors. To demonstrate that AMPA receptors can be recruited to nascent neurexin/neuroligin contacts through lateral diffusion, the mobility of AMPA receptors in the neuronal membrane is monitored by tracking individual quantum dots (QDs) conjugated to antibodies against AMPA receptors. Experiments monitoring the attachment and detachment of Nrx‐coated QDs to measure the rates of neurexin/neuroligin interaction can also be performed. Each of these assays is detailed in this unit. Curr. Protoc. Neurosci. 64:2.19.1‐2.19.30. © 2013 by John Wiley & Sons, Inc.

Keywords: hippocampal neurons; immunostaining; single particle tracking; synaptic adhesion molecules; protein production; quantum dots

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Neurexin Cluster Assay to Induce Postsynaptic Formation
  • Support Protocol 1: Production of Recombinant Neurexin 1β‐Fc
  • Support Protocol 2: Immunostaining of Excitatory and Inhibitory Synapses Induced by Neuroligin
  • Alternate Protocol 1: Live Neurexin Cluster Assay to Induce Postsynaptic Formation
  • Alternate Protocol 2: Use of Neurexin‐Coated Microspheres to Induce Postsynaptic Formation
  • Basic Protocol 2: Quantum Dots to Track Surface‐Diffusing Endogenous and Recombinant AMPA Receptors
  • Support Protocol 3: Production of Anti‐GluA2 Fab
  • Alternate Protocol 3: Quantum Dots to Measure Neurexin/Neuroligin Interaction Kinetics
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Neurexin Cluster Assay to Induce Postsynaptic Formation

  Materials
  • Feeding medium (see recipe)
  • Globulin‐ and protease‐free bovine serum albumin (BSA, Sigma, A3059)
  • Purified recombinant Nrx1β‐Fc (see protocol 2)
  • Goat anti−human Fc antibody (Jackson ImmunoResearch, cat. no. 109‐005‐098) conjugated to Cy5 using a commercial kit (GE Healthcare Life Sciences, Cy5 mono‐reactive dye pack PA25001) according to manufacturer's instructions, stored at −20°C
  • Banker culture of hippocampal neurons expressing HA:Nlg1 (extracellular HA tag fused to Nlg1 sequence) plated on 0.17‐mm glass coverslips (Kaech and Banker, )
  • Primary antibodies:
    • Rat monoclonal anti‐HA antibody (Roche, cat. no. 11867423001)
    • Mouse monoclonal anti‐PSD‐95 antibody (NeuroMab, clone K28/43 75028)
  • Secondary antibodies:
    • Rhodamine‐conjugated goat anti‐mouse (minimal cross‐reaction with rat, Jackson ImmunoResearch, 115‐025‐66)
    • FITC‐conjugated goat anti‐rabbit (minimal cross‐reaction with mouse, Jackson ImmunoResearch, cat. no. 112‐095‐167)
  • Humidified, 37°C, 5% CO 2 incubator
  • Sterile 50‐ml culture flasks
  • 1.5‐ml microcentrifuge tubes
  • Dark incubation chamber
  • Parafilm
  • Tweezers
  • 12‐well tissue culture plate
  • Epifluorescence microscope (e.g., Leica DM R) with 63×/1.32 NA oil‐immersion objective and appropriate filter sets
  • CCD camera (e.g., HQ CoolSnap, Roper Scientific)
  • MetaMorph software (Molecular Devices)

Support Protocol 1: Production of Recombinant Neurexin 1β‐Fc

  Materials
  • Frozen ECCAC HEK‐293 cells in liquid nitrogen container
  • Complete DMEM (see recipe), 37°C
  • 1× phosphate‐buffered saline (PBS; e.g., Euromedex), 37°C
  • 1× trypsin‐EDTA (Fisher, cat. no. 25300‐054), 37°C
  • pcDNA Hygro Nrx1β‐Fc DNA (Scheiffele et al., )
  • Amaxa Cell Line Nucleofactor V Kit (Lonza, cat. no. VCA‐1003)
  • 50 mg/ml hygromycin B (Fisher, cat. no. W3397L)
  • AIM‐V medium (Fisher, cat. no. 31035‐025), 37°C
  • Goat anti−human Fc antibody (Jackson ImmunoResearch, cat. no. 109‐005‐098)
  • Rabbit anti‐goat conjugated to alkaline phosphatase (p.a.r.i.s., BI 4403)
  • SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific, cat. no. 34095)
  • 0.1 M dithiothreitol (DTT, Sigma, cat. no. P2256), 4°C
  • Concentrated HCl or NaOH
  • Milli‐Q water, 4°C
  • Binding buffer: 20 mM sodium phosphate buffer, pH 7, 4°C
  • 1 M Tris⋅Cl, pH 9 ( appendix 2A), 4°C
  • Elution buffer: 100 mM glycine (Sigma, cat. no. G8898), pH 2.7, 4°C
  • 20% (v/v) ethanol, 4°C
  • Colloidal blue staining solution (see recipe)
  • 37°C water bath
  • Sterile 250‐ml culture flasks
  • Humidified, 37°C, 5% CO 2 incubator
  • 15‐ and 50‐ml conical centrifuge tubes
  • Malassez hemocytometer
  • Tabletop centrifuge with rotor compatible with 15‐ and 50‐ml conical tubes
  • Amaxa Nucleofactor 2b device
  • 500‐ml, 0.2‐µm filtration units
  • 1‐ml HiTrap G‐protein HP column (GE Healthcare, cat. no. 17‐0407‐03), 4°C
  • AKTAprime plus chromatography system (GE Healthcare, cat. no. 11‐0013‐13), 4°C
  • pH strips
  • Collection tubes (Dominique Dutscher, cat. no. 110034)
  • Micro BCA Protein Assay Kit (Thermo Scientific, cat. no. 23235)
  • Spectrophotometer with cuvettes
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and western blotting (Gallagher et al., )

Support Protocol 2: Immunostaining of Excitatory and Inhibitory Synapses Induced by Neuroligin

  Materials
  • Fixative solution (see recipe), 37°C
  • Banker culture of hippocampal neurons expressing HA:Nlg1, plated on 0.17‐mm glass coverslips (Kaech and Banker, )
  • 1× phosphate‐buffered saline (PBS; e.g., Euromedex)
  • 50 mM NH 4Cl, freshly prepared from 1 M NH 4Cl stock in 1× PBS
  • Permeabilization solution (see recipe)
  • Blocking solution: 1% BSA (Sigma, cat. no. A7638) in 1× PBS, freshly prepared (do not stir)
  • Primary antibodies:
    • Mouse monoclonal anti‐PSD‐95 (NeuroMab, clone K28/43 75028)
    • Mouse monoclonal anti‐gephyrin (Synaptic Systems, cat. no. 160004)
    • Rabbit polyclonal anti‐synaptotagmin (generous gift from C. Dotti, VIB Center for Biology of Disease, K.U. Leuven)
  • Secondary antibodies:
    • Alexa 568−conjugated goat anti‐mouse (Life Technologies, cat. no. A11004)
    • Alexa 488−conjugated goat anti‐rabbit (Life Technologies, cat. no. A11008)
  • Mowiol anti‐fade mounting medium (see recipe)
  • Clear nail varnish
  • 12‐well tissue culture plate
  • Orbital shaker
  • Dark incubation chamber
  • Parafilm
  • Microscope slides
  • Epifluorescence microscope (e.g., Leica DM R) with 63×/1.32 NA oil‐immersion objective and appropriate filter sets
  • CCD camera (e.g., HQ CoolSnap, Roper Scientific)
  • MetaMorph software (Molecular Devices)

Alternate Protocol 1: Live Neurexin Cluster Assay to Induce Postsynaptic Formation

  • Tyrode solution (see recipe)
  • Banker culture of hippocampal neurons expressing HA:Nlg1 and PSD‐95:GFP (GFP tag inserted between PSD‐95 PDZ2 and PDZ3 domains) plated on 0.17‐mm glass coverslips (Kaech and Banker, )
  • 35‐mm tissue culture dishes

Alternate Protocol 2: Use of Neurexin‐Coated Microspheres to Induce Postsynaptic Formation

  • 4‐µm sulfate latex beads (Invitrogen, cat. no. IDC S37225, 4% w/v)
  • 0.2 M borate buffer (see recipe)
  • Banker cultures of hippocampal neurons expressing HA:Nlg1 and PSD‐95:mCherry (mCherry tag fused at C‐terminal of PSD‐95 sequence) plated on 0.17‐mm glass coverslips (Kaech and Banker, )

Basic Protocol 2: Quantum Dots to Track Surface‐Diffusing Endogenous and Recombinant AMPA Receptors

  Materials
  • Feeding medium (see recipe)
  • Tyrode solution (see recipe)
  • Globulin‐ and protease‐free bovine serum albumin (BSA; Sigma, cat. no. A3059)
  • 1× phosphate‐buffered saline (PBS; e.g., Euromedex)
  • Primary antibodies:
    • Mouse Fab anti‐GluA2 (see protocol 7)
    • Rabbit polyclonal anti‐GluA1 (generous gift from M.D. Ehlers, Pfizer)
    • Rabbit polyclonal anti‐GFP (Life Technologies, cat. no. A‐6455)
  • Anti‐mouse‐ or anti‐rabbit‐conjugated 655‐nm quantum dots (Qdot 655, Life Technologies, cat. no. Q11021MP or Q11421MP)
  • Banker culture of hippocampal neurons expressing the one of the following, plated on 0.17‐mm glass coverslips (Kaech and Banker, ):
    • Homer1c:GFP ± HA:Nlg1 (for study of endogenous AMPARs)
    • Homer1c:dsRed + SEP:GluA1 or SEP:GluA2 ± HA:Nlg1 (for study of recombinant AMPARs)
  • 1.5‐ml microcentrifuge tubes
  • 12‐well tissue culture plate
  • Humidified, 37°C, 5% CO 2 incubator
  • Tweezers
  • Microscope with live microscopy observation chamber (e.g., Nikon Eclipse TE 2000‐U) and:
    • Objective heater (Bioptechs)
    • Air blower (Precision instruments)
    • Mercury lamp
    • 100×/1.4 NA oil‐immersion objective
    • FITC filter set (ex. 482/35, DM 506, em. 536/40; Nikon)
    • HCRed1 filter set (ex. HQ575/50, DM Q610LP, em. HQ640/50; Chroma Technology)
  • EMCCD camera with 20 MHz digitizer (QuantEM 512SC, Photometrics, Roper Scientific)
  • MetaMorph software (Molecular Devices)
  • MATLAB software (Mathworks)

Support Protocol 3: Production of Anti‐GluA2 Fab

  Materials
  • Competent Origami 2(DE3) pLysS cells (Novagen, cat. no. 71346)
  • pFab7‐SP plasmid (Jespersen et al., )
  • LB medium (unit 1.1 or Ficher Bioreagents, cat. no. BP 1426‐2)
  • 100‐mm LB agar plates (unit 1.1 or Euromedex, cat. no. EU0031) containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol
  • LB medium containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol
  • LB medium containing 50 µg/ml ampicillin
  • 1 M IPTG (see recipe)
  • 0.9% (w/v) NaCl, 4°C
  • Lysis buffer (see recipe), freshly prepared
  • 42°C heating block
  • 16°‐37°C orbital shaker
  • 2.8‐liter Ferbach culture flask
  • Spectrophotometer and cuvettes
  • 1‐liter centrifuge bottle assembly, polycarbonate (Beckman Coulter, cat. no. A98812)
  • Avanti J‐26 XP centrifuge with JLA 8.1 and JA 18 rotors (or equivalent)
  • 50‐ml conical centrifuge tube
  • Rotating wheel, 4°C
  • 500‐watt ultrasonic processor (Sonics, cat. no. VC505)
  • 0.2‐µm filter
  • Additional reagents and equipment for purification and quality control (see protocol 2)

Alternate Protocol 3: Quantum Dots to Measure Neurexin/Neuroligin Interaction Kinetics

  • Rat monoclonal anti‐HA (Roche, cat. no. 11867423001)
  • Purified recombinant Nrx1β‐Fc (see protocol 2)
  • Anti‐human‐ or anti‐rat‐conjugated 655‐nm quantum dots (Qdot 655, Life Technologies, cat. no. Q11221MP or Q11621MP)
  • Banker culture of hippocampal neurons expressing HA:Nlg1, plated on 0.17‐mm glass coverslips (Kaech and Banker, )
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Figures

Literature Cited

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