Imaging Nervous System Activity with Voltage‐Sensitive Dyes

Dejan Zecevic1, Maja Djurisic2, Lawrence B. Cohen1, Srdjan Antic1, Matt Wachowiak3, Chun X. Falk3, Michal R. Zochowski1

1 Yale University School of Medicine, New Haven, Connecticut, 2 RedShirtImaging, Fairfield, Connecticut, 3 Warsaw School of Advanced Social Psychology, Warsaw, Poland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.17
DOI:  10.1002/0471142301.ns0617s23
Online Posting Date:  August, 2003
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Abstract

Optical recording with a voltage-sensitive dye is advantageous where membrane potential must be recorded in many sites at once. This unit describes methods for making voltage-sensitive dye measurements on different preparations to study (1) how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and where spikes are initiated; (2) how a nervous system generates a behavior in Aplysia abdominal ganglion; and (3) responses to sensory stimuli and generation of motor output in the vertebrate brain by simultaneous measurement of population signals from many areas. The approach is three-pronged: (1) find the dye with the largest signal-to-noise ratio; (2) reduce extraneous sources of noise; and (3) maximize the number of photons measured to reduce the relative shot noise. A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Intracellular Injection of Voltage-Sensitive Dyes into Neurons in Invertebrate Ganglia
  • Alternate Protocol: Intracellular Injection of Voltage-Sensitive Dyes into Neurons in Mammalian Olfactory Bulb Slice Preparations
  • Basic Protocol 2: Bath Application of Voltage-Sensitive Dyes for Recording Action Potentials from Individual Cell Bodies in Invertebrate and Vertebrate Ganglia: The Aplysia Abdominal Ganglion
  • Basic Protocol 3: Application of Voltage-Sensitive Dyes for Recording Population Signals Using in Vivo Vertebrate Preparations: The Turtle Olfactory Bulb
  • Reagents and Solutions
  • Commentary
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Intracellular Injection of Voltage-Sensitive Dyes into Neurons in Invertebrate Ganglia

 Materials
  • Helix aspersa terrestrial snails (Sea Life Supply)
  • Helix saline (see recipe)
  • 0.5% (w/v) Type IX trypsin (Sigma) in Helix saline: store up to 2 weeks at –20°C (i.e., frozen) in 0.5-ml aliquots
  • 0.5% (w/v) Type II-S trypsin inhibitor (Sigma) in Helix saline: store up to 2 weeks at 4°C in 0.5-ml aliquots
  • Dye solution (see recipe)
  • Dissecting instruments: fine scissors and no. 5 forceps
  • Sylgard-coated chamber
  • Dissecting microscope (e.g., Olympus SZ 6045)
  • Sharp microelectrode (see recipe)
  • Micromanipulator
  • Microelectrode amplifier (e.g., Model - 505A; Warner Instruments)
  • Picospritzer (General Valve)
  • Optical recording device (Fig. 6.17.2):
  •     Excitation-interference filter
  •     Dichroic mirror
  •     Barrier filter (RG610; Schott)
  •     464-element photodiode array data acquisition system (i.e., NeuroPlex; RedShirtImaging) including low-pass (4-pole Bessel) filter and RC filter with a cutoff frequency of 1.7 Hz
  •     Computerized data acquisition system (e.g., Model DAP 3200e/214, Microstar Laboratories)
  •     High-resolution CCD camera with controller (CCD-300-RC; Dage-MTI)
  •     FlashBus frame grabber (Integral Technologies)
  •     NeuroCCD-SM camera system (RedShirtImaging)
  •     Upright compound microscope (Model E600FN; Nikon)
  •     Vibration-isolation table (Model 300-SP-1; Minus-k Technology)
  •     250-W xenon arc-lamp (Model 770× W/T; Opti Quip, Highland Mills)
  •     Microelectrode amplifier (e.g., Model - 505A; Warner Instruments)
  •     Axoclamp 200B amplifier
  •     Stimulator with stimulus isolation unit (Model A310; WPI)
  •     Oscilloscope, (Model TEK5103N/02/D11; Tektronix)
  •     Micromanipulator for patch-electrodes (Model MP-285; Sutter Instrument)
  •     Micromanipulator for stimulating metal electrodes (Model NMN-21; Narishige)
  • Additional reagents and equipment for backfilling microelectrodes (unit 6.3)

Alternate Protocol: Intracellular Injection of Voltage-Sensitive Dyes into Neurons in Mammalian Olfactory Bulb Slice Preparations

 Additional Materials (also see Basic Protocol 1)
  • 18- to 25-day-old Wistar rats
  • Halothane
  • Vertebrate extracellular solution (see recipe)
  • Intracellular solution (see recipe)
  • Rodent guillotine
  • 5- to 7-M patch pipets (unit 6.3)
  • Infrared differential interference contrast (IR-DIC) video microscope
  • Additional reagents and equipment for anesthetizing rodents (appendix 4B), vibratome sectioning (unit 1.1), back-filling microelectrodes (unit 6.3), and patch-clamping (unit 6.6)

NOTE: All protocols using live vertebrates must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 2: Bath Application of Voltage-Sensitive Dyes for Recording Action Potentials from Individual Cell Bodies in Invertebrate and Vertebrate Ganglia: The Aplysia Abdominal Ganglion

 Materials
  • 5- to 20-g Aplysia californica (Marinus or University of Miami Aplysia Resource Facility)
  • Isotonic (345 mM) MgCl2
  • 50:50 artificial sea water (see recipe)/isotonic MgCl2
  • 0.15 mg/ml RH 155 dye (Nippon Kankoh-Shikiso Kenkyusho) in artificial sea water
  • Artificial sea water with and without low calcium and high magnesium (see recipes)
  • Dissection equipment: fine scissors, Dumont no. 5 forceps, pins
  • Lucite chamber with separate compartments
  • Petroleum jelly (e.g., Vaseline)
  • Computer-controlled galvanometer motor
  • 0.6-mm-diameter glass rod with rounded tip
  • 25× 0.4-NA microscope objective
  • 464-element photodiode array camera system (i.e., NeuroPlex; RedShirtImaging)
  • Videotape recorder
  • Threshold discriminator
  • Additional reagents and equipment for dissection of Aplysia siphon, abdominal ganglion, and gill (Kupfermann, 1971)

NOTE: Contact Dr. L. Cohen at Yale University (lawrence.cohen@yale.edu) for more information concerning construction of the apparatus.

Basic Protocol 3: Application of Voltage-Sensitive Dyes for Recording Population Signals Using in Vivo Vertebrate Preparations: The Turtle Olfactory Bulb

 Materials
  • Terepene carolina and ornata turtles (Charles D. Sullivan Co.)
  • 1% (w/v) lidocaine (Sigma) in turtle saline
  • Tubocurarine (Sigma)
  • Cyanoacrylate glue (e.g., Krazy Glue)
  • Epoxy
  • 0.01 to 0.2 mg/ml RH 414 (Molecular Probes; Fig. 6.17.8D) in turtle saline
     FigureFigure 6.17.8 (A) A schematic diagram of the olfactometer. Compressed air containing 1% CO2 and which has been cleaned and desiccated is used as the carrier gas. This is mixed with room air saturated with odorant vapor in the odor applicator. Flow-rates of the air and odorant vapor are controlled using a flowmeter and syringe pump, respectively. The odor-applicator has two barrels; the outer one is normally under suction to remove the odor. Turning off the suction to the outer barrel releases odorant from the end of the applicator. The output of the odor from the applicator is monitored by measuring the CO2 of the carrier gas using a CO2 detector. (B) Time course of the odor output from the olfactometer measured by monitoring the CO2 in the carrier gas. The upper trace shows the time-course of the command pulse delivered to the suction solenoid of the outer barrel of the odor applicator. The lower trace is the output of the CO2 detector probe. The inlet of the probe was placed near the mouth of the odor applicator. There is a delay of ~100 ms between the command pulse and the arrival of the pulse at the CO2 detector. The odor-pulse is approximately square-shaped. (C) Schematic diagram of the optical imaging apparatus. The olfactory bulb was illuminated using a 100-W tungsten-halogen lamp. The incident light passed through a heat filter and a 520 ± 45-nm band-pass interference filter and was reflected onto the preparation by a 580-nm long-pass dichroic mirror. The image of the preparation is formed by a 25-mm, 0.95-f camera lens onto a 464-element photodiode array after passing through a 610-nm long-pass secondary filter. The secondary filter is needed to block reflected incident wavelengths that are transmitted by the dichroic mirror. The output of each element of the array was amplified by a set of 464 amplifiers. The amplifier outputs were multiplexed, digitized, and stored in a computer. (D) The chemical structure of the styryl dye, RH 414, used in these experiments. This dye was obtained as dibromide salt from Molecular Probes.
  • Turtle saline (see recipe)
  • Odorant (e.g., cineole)
  • Dremel tool with small round bit
  • Dumont no. 5 forceps
  • Polyethylene tubing with outer diameter of 2 mm and an inner diameter of 1 mm
  • Flexible plastic
  • Tape
  • Olfactometer (Kauer and Moulton, 1974; Fig. 6.17.8A)
  • CO2 detector (e.g., Medical Gas Analyzer LB-2; Beckman)
  • Optical measuring and recording device (Fig. 6.17.8C):
  •     4× macroscope (RedShirtImaging) with 25-mm focal length, 0.95-f-stop, C-mount camera lens
  •     100-W tungsten-halogen lamp (Osram)
  •     520 ± 45-nm excitation filter
  •     580-nm long-pass dichroic mirror (Omega Optical)
  •     464-element photodiode array (NeuroPlex; RedShirtImaging)
  •     RG610 long-pass filter (Schott Glass Technologies)
  •     CCD camera (e.g., RC300; DAGE-MTI)

NOTE: All protocols using vertebrates must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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Figures

  •  FigureFigure 6.17.1 Schematic drawings of the three preparations used as examples. (A) An individual cortical hippocampal CA1 pyramidal cell. Each pixel of a camera receives light from a small part of the dendrite, axon, or cell body. An optical measurement of membrane potential would provide important information about how the neuron converts its synaptic input into its spike output. (B) Drawing of a slice through an invertebrate ganglion with its cell bodies in a cortex around the outside and neuropil in the middle. Here each detector would receive light from one or a small number of cell bodies. A voltage-sensitive-dye measurement of spike activity while the ganglion is generating a behavior would provide important information about how the behavior is generated. (C) A vertebrate brain with the superimposed 464-element photodiode array. In this circumstance, each pixel of the array would receive light from thousands of cells and processes. The signal would be the population average of the change in membrane potential in those cells and processes. The image of the hippocampal neuron was taken from Mainen et al. (1996).
    View Image
  •  FigureFigure 6.17.2 The schematic of main components of the apparatus for voltage imaging of electrical activity of individual neurons in invertebrate ganglia and vertebrate brain slices (see Commentary for a discussion of individual components).
    View Image
  •  FigureFigure 6.17.3 Voltage-sensitive dye recording from an invertebrate (Helix pomatia) neuron. (A) Raw optical recordings of fractional fluorescence signals (F/F) associated with an 85-mV action potential from elements of the photodiode array positioned over the fluorescent CCD image of the axonal arborizations of a metacerebral cell in situ, stained with the voltage-sensitive dye JPW 1114. (B) Synaptically-evoked action potential recorded by a microelectrode in the soma. (C) Superimposed recordings from individual detectors from different locations as indicated in panel A. (D) Color-coded representation of the spatial and temporal dynamics of the synaptically-evoked spike. The peak of the action potential, marked by an arrow, corresponds to red color. Individual frames are separated by 0.6 msec.
    View Image
  •  FigureFigure 6.17.4 Voltage-sensitive dye recording from a neuron from the vertebrate CNS. (A) olfactory bulb slice. (B) Mitral cell stained with a voltage-sensitive dye by intracellular deposition. (C) The image of the stained cell as projected onto a CCD camera. (D) Optical signals spatially averaged from three different regions (red, green, and blue) and compared on an expanded time scale. (E) Consecutive color-coded images separated by 0.37 msec (peak of the action potential; corresponds to red color).
    View Image
  •  FigureFigure 6.17.5 Schematic diagram of the apparatus. Light from a tungsten-halogen lamp (light source) is passed through a 720 ± 25 nm interference filter and focused on the preparation using a modification of Kohler illumination (Inoue, 1986). The condenser iris is opened so that the condenser numerical aperture equals the objective numerical aperture. A 464-element photodiode array is placed at the plane where the objective forms the real, inverted image. With this optical arrangement, the image of a three dimensional ganglion is formed on a two-dimensional array. The microscope objective is focused near the middle of the ganglion in an attempt to obtain an equal signal-to-noise ratio from cell bodies on both the top and bottom surfaces of the ganglion. Since the position of the neurons in the Z axis (optical path) was unknown in this study, no attempt was made to determine if any of the individual neurons whose activity was detected optically could be identified as previously studied neurons. The structure of the dye used for these experiments is shown on the lower right. Reprinted with permission from Wu et al. (1998).
    View Image
  •  FigureFigure 6.17.6 (on left) Raster diagram of the action-potential activity recorded optically from an Aplysia abdominal ganglion during a gill-withdrawal reflex. The 0.5-sec touch to the siphon began at the time of the line labeled “stim.” In this recording, activity in 135 neurons was measured; however, this recording is incomplete. The actual number of active neurons was estimated to be between 250 and 300. Most neurons are activated by the touch but one, #4334 (~ frac13 down from the top), was inhibited. This inhibition was seen in repeated trials in this preparation. The curve at the bottom is a recording of the gill movement. Reprinted with permission from Wu et al. (1998).
    View Image
  •  FigureFigure 6.17.7 Parent structures for RH and JPW dyes. Results of perfusion studies are given in Table 6.17.1.
    View Image
  •  FigureFigure 6.17.8 (A) A schematic diagram of the olfactometer. Compressed air containing 1% CO2 and which has been cleaned and desiccated is used as the carrier gas. This is mixed with room air saturated with odorant vapor in the odor applicator. Flow-rates of the air and odorant vapor are controlled using a flowmeter and syringe pump, respectively. The odor-applicator has two barrels; the outer one is normally under suction to remove the odor. Turning off the suction to the outer barrel releases odorant from the end of the applicator. The output of the odor from the applicator is monitored by measuring the CO2 of the carrier gas using a CO2 detector. (B) Time course of the odor output from the olfactometer measured by monitoring the CO2 in the carrier gas. The upper trace shows the time-course of the command pulse delivered to the suction solenoid of the outer barrel of the odor applicator. The lower trace is the output of the CO2 detector probe. The inlet of the probe was placed near the mouth of the odor applicator. There is a delay of ~100 ms between the command pulse and the arrival of the pulse at the CO2 detector. The odor-pulse is approximately square-shaped. (C) Schematic diagram of the optical imaging apparatus. The olfactory bulb was illuminated using a 100-W tungsten-halogen lamp. The incident light passed through a heat filter and a 520 ± 45-nm band-pass interference filter and was reflected onto the preparation by a 580-nm long-pass dichroic mirror. The image of the preparation is formed by a 25-mm, 0.95-f camera lens onto a 464-element photodiode array after passing through a 610-nm long-pass secondary filter. The secondary filter is needed to block reflected incident wavelengths that are transmitted by the dichroic mirror. The output of each element of the array was amplified by a set of 464 amplifiers. The amplifier outputs were multiplexed, digitized, and stored in a computer. (D) The chemical structure of the styryl dye, RH 414, used in these experiments. This dye was obtained as dibromide salt from Molecular Probes.
    View Image
  •  FigureFigure 6.17.9 Examples of four different chromophores that have been used to monitor membrane potential. The merocyanine dye, XVII (WW 375), and the oxonol dye, RH 155, are commercially available as NK2495 and NK3041, respectively, from Nippon Kankoh-Shikiso Kenkyusho. The oxonol, XXV (WW 781) and styryl, di-4-ANEPPS, are available commercially as dye R-1114 and D-1199 from Molecular Probes. Reprinted with permission from Wu et al. (1998).
    View Image
  •  FigureFigure 6.17.10 Plots resulting from using a table of random numbers to distribute 20 photons (A) or 200 photons (B) into 20 time bins. The result illustrates the fact that when more photons are measured relative noise is decreased. On the right, the signal-to-noise ratio is measured for the two results. The ratio of the two signal-to-noise ratios was 2.8. This is close to the ratio predicted by the relationship that the signal-to-noise ratio is proportional to the square root of the measured intensity. Reprinted with permission from Wu et al. (1998).
    View Image
  •  FigureFigure 6.17.11 The ratio of light intensity divided by the noise in the measurement as a function of light intensity in photons/millisecond/0.2% of the object plane. The theoretical optimum signal-to-noise ratio (dotted line) is the shot-noise limit. In addition, two camera systems are shown, a photodiode array with 464 pixels (solid lines) and a cooled, back-illuminated, 2-kHz-frame-rate, 80 × 80–pixel CCD camera (dashed lines). The photodiode array provides an optimal signal-to-noise ratio at higher intensities while the CCD camera is better at lower intensities. The approximate light intensity per detector from fluorescence measurements from a single neuron, from fluorescence measurements from a slice or in vivo preparation, and absorption measurements from a ganglion or a slice are indicated along the x axis. The signal-to-noise ratio for the photodiode array falls away from the ideal at high intensities (A) because of extraneous noise and at low intensities (C) because of dark noise. The lower dark noise of the cooled CCD allows it to function at the shot-noise limit at lower intensities until read noise dominates (D). The CCD camera saturates at intensities above 5 × 106 photons/msec/0.2% of the object plane.
    View Image
  •  FigureFigure 6.17.12 Effects of focus and scattering on the distribution of light from a point source onto the array, with the individual trace of each detector shown. A 10× 0.4-NA objective and Kohler illumination with light at 750 nm was used and a 40-µm pinhole in aluminum foil was placed in the object plane. The recording gains were adjusted so the largest signal in each of the three trials would be approximately the same size in the figure. (A) The pinhole was covered with saline and was in focus. More than 90% of the light fell on one detector. (B) The stage was moved downward by 500 µm. Light from the out-of-focus pinhole was now seen on several detectors. (C) The pinhole was in focus but covered by a 500-µm slice of salamander olfactory bulb. Again the light from the pinhole was spread over several detectors. Reprinted with permission from Wu et al. (1998).
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  •  FigureFigure 6.17.13 Comparison of photodiode outputs (top) and the analyzed results from that data, neuron activity (bottom). The outputs of seven detectors, indicated in the drawing on the top left, from a portion of an 8-sec recording are shown on the top right. The numbers to the left of each trace indicate the photodetector number. Examination of the photodiode outputs indicated that activity in four neurons would account for all of the large optical signals. The activity of these four neurons is indicated in a raster diagram on the bottom right. The schematic drawing on the bottom left indicates the diodes on which the signals from the four neurons can be detected. Instances of the signals from these four neurons are indicated by the numbers on the traces on the top left. The length of the vertical line to the right of the traces represents the stated value of the average change in intensity divided by the resting intensity (I/I) for the seven traces. In addition to the high-frequency filtering provided by the amplifier system, the raw data was smoothed with two passes of a 1-2-1 digital smoothing routine. Reprinted with permission from Wu et al. (1998).
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  •  FigureFigure 6.17.14 Simultaneous optical recordings from seven different areas of an olfactory bulb. An image of the olfactory bulb is shown on the left. Signals from seven selected pixels are shown on the right. The positions of these pixels are labeled with squares and numbers on the image of the bulb. All seven signals have a filtered version of the DC signal at the time indicated by the bar-labeled DC. The oscillation in the rostral region has a high frequency and relatively long latency and duration (detectors 1 and 2). The oscillation from the middle region has a high frequency, short latency, and short duration (detector 4). The oscillation from the caudal region has a lower frequency and the longest latency (detector 7). The signal from detectors between these regions (3, 5, and 6) appear to contain a mixture of two components. The horizontal line labeled “10% cineole” indicates the time of the command pulse to the odor solenoid. The data was filtered using high-pass digital RC (5 Hz) and low-pass Gaussian (30 Hz) filters.
    View Image

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