All‐Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons

Christopher A. Werley1, Ted Brookings1, Hansini Upadhyay1, Luis A. Williams1, Owen B. McManus1, Graham T. Dempsey1

1 Q‐State Biosciences, Cambridge, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.20
DOI:  10.1002/cpph.25
Online Posting Date:  September, 2017
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A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low‐throughput, invasive methods or lack cell‐specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single‐cell sensitivity and millisecond temporal resolution. Neuronal activity is elicited using blue light activation of the channelrhodopsin variant ‘CheRiff’. Action potentials and synaptic signals are measured with ‘QuasAr’, a rapid and sensitive voltage‐indicating protein with near‐infrared fluorescence that scales proportionately with transmembrane potential. This integrated technology of optical stimulation and recording of electrical signals enables investigation of neuronal electrical function with unprecedented scale and precision. © 2017 by John Wiley & Sons, Inc.

Keywords: CheRiff; disease modeling; induced pluripotent stem cell; optogenetics; Optopatch; Optical electrophysiology; QuasAr; voltage indicator

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Production of Lentivirus Encoding Optopatch Components
  • Basic Protocol 2: Culture and Transduction of Human Differentiated Neurons (CDI iCELL Neurons)
  • Basic Protocol 3: Culture and Transduction of Primary Rat Hippocampal Neurons
  • Basic Protocol 4: Optopatch Imaging of Neurons
  • Basic Protocol 5: Extraction of Neuronal Firing Properties From High‐Speed Video
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Production of Lentivirus Encoding Optopatch Components

  • HEK293T cells (ATCC #CRL3216)
  • 10% FBS medium (see recipe)
  • Phosphate‐buffered saline (ThermoFisher Scientific, cat. no. 10010‐049)
  • Freezing medium: 20% dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. D2650‐100 ml) in fetal bovine serum (FBS; HyClone, cat. no. SH30071.02HI)
  • Liquid N 2
  • Lentiviral plasmid with promoter and Optopatch gene
  • Viral packaging mix containing plasmids for PsPAX2 and PMD2.G (contains VSVG gene), supplied as 250 μg in a 0.5 μg/ml solution (Cellecta, cat. no. CPCP‐K2A)
  • Opti‐MEM reduced serum medium (ThermoFisher Scientific, cat. no. 31985‐070)
  • Polyethylenimine (PEI) MAX40000 transfection reagent (see recipe)
  • Lenti‐X Concentrator (Takara Clontech, cat. no. 631231)
  • Neurobasal medium (ThermoFisher Scientific, cat. no. 10888‐022)
  • Lenti‐X GoStix (Takara Clontech, cat. no. 631243)
  • Lenti‐X qRT‐PCR Titration Kit (Takara Clontech, cat. no. 631235)
  • 10‐cm (diameter) tissue culture dishes (Corning, cat. no. 353003)
  • 15‐ml (Corning, cat. no. 352196) and 50‐ml (Corning, cat. no. 352050) conical tubes
  • 1.8‐ml cryovials (Thermo Scientific Nunc, cat. no. 377267)
  • Liquid nitrogen storage unit
  • 15‐cm (diameter) tissue culture dishes (Corning, cat. no. 352196)
  • Epifluorescence microscope equipped with fluorescence objectives and filter cubes matched to the fused fluorescent protein (e.g., Semrock DAPI‐11LP‐A‐000, GFP‐4050B‐000, LED‐TRITC‐A‐000)
  • Centrifuge
  • 0.45 μm Steriflip‐HV (EMD Millipore, cat. no. SE1M003M00)
  • 0.6‐ml sterile tubes (ThermoFisher Scientific, cat. no. 3449)

Basic Protocol 2: Culture and Transduction of Human Differentiated Neurons (CDI iCELL Neurons)

  • Poly‐D‐lysine (Sigma, cat. no. P6407‐5MG)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐049)
  • 20 µg/ml laminin (Life Technologies, cat. no. 23017‐015)
  • iCell Neurons (Cellular Dynamics Inc. CDI, cat. no. NRC‐100‐010‐001)
  • iCell Neuron maintenance medium (CDI, cat. no. NRM‐100‐121‐001)
  • iCell Neuron maintenance medium supplement (CDI, cat. no. NRM‐100‐031‐001)
  • Lentivirus stocks ( protocol 1)
  • 0.25% trypsin‐EDTA (Sigma‐Aldrich, cat. no. T4049‐500 ML)
  • Primary glial cells (see text above step 17)
  • Glial medium (see recipe)
  • Glass‐bottomed dishes (MatTek, cat. no. P35G‐1.5‐10‐C)
  • 15‐ml (Corning, cat. no. 352196) and 50‐ml (Corning, cat. no. 352050) conical tubes
  • Poly‐D‐lysine‐coated T‐25 flasks (Corning, cat. no. 356536)
  • Centrifuge with swinging‐bucket rotors

Basic Protocol 3: Culture and Transduction of Primary Rat Hippocampal Neurons

  • Papain enzyme with Hibernate E‐CA media (Brain Bits, cat. no. PAP/HE)
  • E18 rat hippocampus tissue (BrainBits)
  • NbActiv1 plating medium (BrainBits, cat. no. NbActiv1 500)
  • Rat neuronal culture feeding medium (see recipe)
  • Lentivirus stocks ( protocol 1)
  • Primary glial cells (see protocol 2)
  • Trans‐retinal (Sigma‐Aldrich, cat. no. R2500‐25mg)
  • Poly‐D‐lysine‐ and laminin‐coated (see protocol 2, steps 1 to 3) glass‐bottom dishes (MatTek, cat. no. P35G‐1.5‐10‐C)
  • 15‐ml conical centrifuge tubes (Corning, cat. no. 352196)
  • Additional reagents and equipment for basic cell culture techniques including counting cells (Phelan & May, )

Basic Protocol 4: Optopatch Imaging of Neurons

  • Neurons transfected with Optopatch (see protocols above)
  • TetraSpeck beads (ThermoFisher, cat. no. T7279)
  • Tyrode's imaging buffer with synaptic blockers
  • Test compounds
  • Custom microscope comprising:
  • Fluorescence sensor—QuasAr is 50× to 100× dimmer than EGFP and requires 500‐ to 1000‐Hz frame rates for recording neuronal action potentials. These technical demands necessitate careful microscope design, the key components of which are as follows.
  • Camera—High sensitivity and high speed are required, limiting practical selections to scientific CMOS (sCMOS) or electron‐multiplied CCD (EMCCD) cameras. We selected the Hamamatsu ORCA‐Flash4.0 sCMOS camera in rolling shutter mode, which offers an excellent combination of high frame rate and low noise.
  • Microscope objective— As efficient collection of light is critical, the highest numerical aperture (NA) oil‐immersion lenses are needed. A 60× Olympus APON 60XOTIRF objective is recommended. It has an NA of 1.49 and enables grazing incidence near total internal reflection (TIR) illumination to reduce background autofluorescence.
  • Red excitation light— A high‐power laser is needed for recordings. A 140‐mW Coherent OBIS 637 LX laser with analog intensity control can be used for this purpose.
  • Blue stimulation light—While the blue light (488 nm) used to stimulate the CheRiff need not be too intense it must be modulated with sub‐millisecond temporal resolution for full flexibility. A 50‐mW Coherent OBIS 488 LX laser with an analog intensity control is recommended.
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Key Reference
  Hochbaum, D. R. et al. 2014. See above.
  Introduces the Optopatch system for the first time, with control experiments described in the supplement.
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