Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Summary

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Abstract

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (V_m-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes ². Many aspects of the initial technology have been continuously improved over several decades ^{3, 5, 11}. Additionally, previous work documented two essential characteristics of V_m-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; ^{10, 14, 16}). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible ^{4, 7}. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects ^{4, 6, 12, 13}. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the V_m-imaging technique. The current sensitivity permits multiple site optical recordings of V_m transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

Protocol

1. Equipment Setup

Step 1.1. Imaging setup

The key to recording voltage sensitive dye signals is appropriate setup design. We use an upright microscope (BX51WI Olympus or Zeiss AxioExaminer) equipped with three cameras. The setup is designed for illuminating individual neurons in brain slices by excitation light in epi-fluorescence, wide-field microscopy mode using either Nikon 60X/1.0 NA or Zeiss 63X/1.0 NA water dipping objectives. Our microscopes are bolted to a vibration isolation table and equipped with motorized movable stages. Each microscope is equipped with three camera ports. One camera port has a standard high spatial resolution CCD camera for infrared DIC video-microscopy (IR-1000, Dage MTI, USA). The second camera port has a fast data acquisition camera (up to 20 kHz frame rate) with relatively low spatial resolution (80 x 80 pixels) but outstanding dynamic range (14 bits) and exceptionally low read noise (NeuroCCD-SM, RedShirtImaging LLC, Decatur, GA). The third camera port has a CCD camera with high spatial resolution (PixelFly, 1392x1024 pixels; PCO AG, Germany) mounted on a spinning-disc confocal scanner (CSU-10, Yokogawa, Japan) used to collect z-stacks of confocal images for detailed morphological reconstruction of the stained cell. A frequency-doubled diode-pumped Nd:YVO4 continuous wave laser (400 mW) emitting at 532 nm (MLL-III/400 mW; CNI, Changchun, China) is the source of excitation light. The 2 mm diameter laser beam gated by a shutter (LS6; Vincent Associates) is directed to a light guide coupled to the microscope via a single-port epifluorescence condenser (TILL Photonics GmbH, Gräfelfing, Germany) designed to overfill the back aperture of the objective and provide near uniform illumination of the object plane. The laser light is used in place of a conventional Xenon arc-lamp to maximize the sensitivity of V_m-imaging by: (1) using a monochromatic excitation light at the red wing of the absorption spectrum to maximize V_m sensitivity of the dye ^{9, 10} and (2) increasing the intensity of the excitation light beyond the level that can be achieved by an arc-lamp. The excitation light was reflected to the preparation by a dichroic mirror with a central wavelength of 560 nm and the recorded fluorescence light was passed through a 610 nm barrier filter (a Schott RG610). The combined effect of an increase in light intensity and the use of near optimal monochromatic excitation wavelengths was a dramatic improvement in the sensitivity of voltage imaging by a factor of about 50 compared to previous measurements ⁶.

Step 1.2. Adjust for uniform illumination

Use a fluorescence slide standard (green excitation/red emission). Insert appropriate neutral density filters in the laser beam path so that the CCD is not saturated. Focus the objective on the surface of the slide. Adjust the position of the receiving end of the quartz light guide in front of the laser launcher objective, and adjust the position of the output end of the light guide attached to the microscope using appropriate actuators on the epi-fluorescence condenser to achieve centered and uniform illumination of the field of view.

Step 1.3. Determine vibrational noise

Place a small black ink mark on the surface of the fluorescence slide. Record light intensity with NeuroCCD in the continuous recording mode. Focus the objective on a dark edge of the black ink mark. Record the light intensity for about 100 msec at 2 kHz frame rate. Display the spatial average of the fractional light intensity traces (ΔF/F) from ~ 20 pixels receiving light from the uniformly illuminated area and from ~20 pixels along the edge of the ink mark. The excess noise in recordings from pixels with high contrast edges reflects the vibrational noise in the system.

Step 1.4. Vibration isolation

It is mandatory to reduce vibrations by using a vibration-isolation table to a level below the shot noise at light intensities comparable to experimental recording conditions. Adjust the vibration isolation table until the vibration noise in the light intensity from pixels covering the sharp edge of the image is negligible. None of the equipment with moving parts (mechanical shutters, fans) can be mounted on the table. The cables from the equipment on the table attached to other components that are not isolated from vibration must be loose so that they do not transmit mechanical vibrations to the microscope.

2. Selection of an Appropriate Neuron for V_m-Imaging

Step 2.1. Selection of neurons

Make brain slices according to standard procedures. Use a mouse line expressing EGFP in individual nerve cells of interest. With a spinning disc confocal system, visualize EGFP labeled neurons in the slice. Select neurons for V_m-imaging with intact dendritic/axonal trees, and with processes running parallel and close to the surface of the slice. This cannot be accomplished in wild-type mice because axons and thin dendrites, for the most part, are not visible in DIC microscopy mode.

3. Loading Neurons with Voltage-sensitive Dye

Step 3.1. Filling of the patch pipette

Fill glass patch pipettes from the tip with dye-free intracellular solution by applying negative pressure for about 15 s up to 2/3 of the electrode taper. The dye free solution in the tip is necessary to prevent the spill of dye onto the slice which increases the background fluorescence and dramatically reduces signal to noise ratio. Back-fill the electrode with the solution containing the membrane impermeant voltage sensitive dye JPW3028 dissolved in intracellular solution (0.8 mM).

JPW3028, the most successful voltage probe for intracellular application, is a doubly positively charged analogue of the ANEP series of lipophilic styryl dyes that is still sufficiently water soluble to be used for microinjection. The di-ethyl analog of this dye has practically identical characteristics (including voltage-sensitivity) and is commercially available as JPW1114 (see Table 1). We prepare 20 mM stock solution in distilled H₂O. 50 μl aliquots of the stock solution are kept frozen at -20 °C. For the final dye concentration of 0.8 mM, 2 μl of the stock solution is dissolved in 50 μl of intracellular solution on the day of the experiment. The stock dye solution is stable and can be kept at room temperature for several months. Thus, we keep one 50 μl aliquot at room temperature until it is used up.

Step 3.2. Establish a giga-seal quickly

Patch a previously selected neuron and allow free diffusion of the dye from a somatic patch pipette to the soma in the whole-cell configuration for 20-45 min. It is essential to obtain a seal as fast as possible so that the amount of dye-free solution in the tip can be kept small. Start by practicing fast patching without the dye in the electrode. Next, practice patching and loading neurons with the dye with the goal of using a minimal amount of dye-free solution in the tip without contaminating the surrounding tissue. With practice, it is possible to establish a seal within 1-2 min.

Step 3.3. Monitor the level of staining

During dye diffusion in the whole-cell configuration, monitor the physiological state of the neuron by recording evoked action potentials in the current-clamp mode. Additionally, monitor the amount of staining by recording the resting light intensity (RLI) from the cell soma at a frame rate of 2 kHz and at a fraction of full light intensity adjusted with neutral density filters (we use 0.04% of the laser light intensity from a 400 mW laser). Continue the dye loading process until the action potential starts to broaden, usually after 20-40 minutes, depending on the electrode size and resistance.

The broadening of the spike is completely reversible and most likely due to capacitive load effect of the saturated concentration of the dye in the somatic membrane. The waveform of the spike is fully restored after the dye concentration is equilibrated throughout the neuron.

Step 3.4. Remove the dye electrode

At the end of staining period, carefully pull the patch pipette away from the soma in voltage clamp configuration ensuring that the transition from whole-cell to outside-out patch configuration is attained in the process.

Step 3.5. Wait for dye diffusion

Incubate the slice for an additional 1.5-2 hr at room temperature to allow for the voltage-sensitive dye to spread into neuronal processes. After a significant amount of dye diffuses away from the soma into dendritic and axonal processes, the waveform of the AP is completely restored.

4. Optical Recording

Step 4.1. Select a cellular compartment for imaging

Locate the soma of the stained neuron under low light level fluorescence and re-patch the neuron with a standard (no dye) patch electrode under DIC. Visualize neuronal processes under low light level at a frame rate of 10-40 Hz in the continuous recording mode of the CCD for voltage-imaging. Reduce the light level with neutral density filters to the minimum required to visualize the object of interest. We use 0.01% of the 400 mW laser intensity during positioning of the stained neuron. Using an X-Y stage, position the neuronal process of interest in the middle of the imaging area. Protect the soma from the excitation light using the partially closed field stop iris of the microscope. Shielding the soma from high intensity excitation light will significantly reduce the photodynamic damage during recording.

Step 4.2 Record optical signals related to membrane potential transients

Record optical signals related to backpropagated APs in individual dendritic branches. Record optical signals related to APs in the axon. Record optical signals related to backpropagating APs in dendritic spines. Use frame rates appropriate for accurate reconstruction of signal waveform and keep the recording periods and exposure to high intensity excitation light as short as possible to minimize dye bleaching and photodynamic damage. For example, examining the sequence of initiation and propagation of single action potentials in the axon requires recording periods of 5-10 msec. The excitation light intensity during recording is a compromise between the signal-to-noise ratio on the one hand and the degree of dye bleaching and photodynamic damage on the other hand. We use 100% of intensity of the 400 mW laser in recording from long sections of axons when an area of 300 μm in diameter is illuminated. When the excitation light is focused to a 30 μm diameter area for dendritic spine imaging, we use 10-25% of the laser light intensity. The required duration of recording is an important determinant of the optimal excitation light intensity; shorter recording periods allow higher light intensities. The optimal illumination intensity is best determined empirically for each preparation and measurement settings.

5. Data Analysis

Step 5.1. Correct the raw data for known errors

The analysis and display of data were carried out using the NeuroPlex program (RedShirtImaging) written in IDL (ITT Visual Information Solutions, Boulder, Colorado) and custom Visual Basic routines. Under the conditions of low light levels, the background fluorescence becomes a significant determinant of the ΔF/F signal size. The raw data were first corrected for this effect by subtracting the average background fluorescence intensity determined from an unstained area on the slice. Subsequently, the signal alignment software was used to correct for temporal jitter in AP initiation as well as for possible small movements of the preparation during averaging. In the temporal domain, AP signals were aligned by cross correlation of the electrically recorded APs in each trial to the reference signal acquired at the start of averaging (Figure 1B). In the spatial domain, images were aligned in two dimensions offline by image cross-correlation to compensate for possible small lateral movements of the preparation. The correct focus of the image in the z-dimension was verified before each individual trial; small adjustments were often necessary. The spatially and temporally aligned signals were averaged as shown in Figure 1B. Slow changes in light intensity due to bleaching of the dye were corrected by dividing the data by an appropriate dual exponential function derived from the recording trials with no stimulation (Figure 1B). The waveform of the AP signal was reconstructed from a set of data points using Cubic Spline Interpolation, a piecewise continuous curve passing through each data point. To confirm that the voltage-sensitive dye signal tracks membrane potential without significant distortion at the millisecond time scale, the electrical AP signal from the soma was compared to the optical AP signal from the adjacent axon hillock as shown in Figure 3B. The two signals superimpose very closely, allowing for the shot noise in optical recording.

Figure 1. Data analysis (Animation). (A) Upper panel: high resolution confocal image of a stained neuron with axon in recording position. Recording electrode attached to soma and extracellular stimulating electrode next to basal dendrite shown schematically. Action potentials evoked by extracellular, synaptic stimulation. Lower panel: low spatial resolution fluorescence image of the axon obtained by CCD used for V_m imaging. (B) Electrode recordings from soma (black traces), optical recordings from AIS (red traces), and from node of Ranvier (green traces). Top traces: raw data from 9 trials showing temporal jitter in AP initiation. Second row of traces: temporally aligned signals. Third row of traces: averaged signal. Fourth row of traces: bleach correction. Bottom traces: cubic spline interpolation with one pass of temporal smoothing.

Results

Successful confocal microscopy should allow clear identification of intact neuronal processes which are close to the surface of the slice and located in one plane of focus. The selection of nerve cells which are appropriate for voltage imaging prior to voltage-sensitive dye loading is critical. An example of confocal images of L5 pyramidal neurons expressing EGFP in a cortical slice (Crym transgenic mouse line) is shown in Figure 2. Axons of individual neurons are clearly visible. Cells with long intact ...

Discussion

This article describes a voltage-sensitive dye recording method for monitoring electrical activity of individual neurons with sub-micrometer and sub-millisecond spatiotemporal resolution. Laser excitation at near-optimal wavelength (regarding signal size) improved the sensitivity of recording by a factor of ~50 over previous approaches. The current sensitivity enables monitoring electrical signals from all parts of individual neurons, including dendrites, axons, axon collaterals and axon terminals as well as individual d...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Setup components
Upright Microscope	Olympus Inc.	BX51WI	With three camera ports
Motorized Movable Stage	Siskiyou	MXOPi.2
Epi-fluorescence Condenser for Olympus BX51	TILL Photonics	0000-560-11659
Upright Microscope	Carl Zeiss, LLC	AxioExaminer D1	With three camera ports
Motorized Top Plate	Scientifica Limited	MMBP
Epi-fluorescence Condenser for Zeiss Axi–xaminer	TILL Photonics
Data Acquisition Camera	RedShirtImaging LLC	NeuroCCD-SM	High speed, low read noise
CCD for IR-DIC	Dage-MTI	IR-1000
Spinning-Disc Confocal Scanner	Yokogawa	CSU-10
High Spatial Resolution CCD on Confocal Scanner	PCO AG	PixelFly	1392x1024 pixels
DPSS CW Laser (532 Nm)	CNI Optoelectronics Tech. Co., Ltd	MLL-III-532 400mW	Excitation light source
Multi-Mode Fiber Launcher	Siskiyou	SM-CFT
Light Guide	TILL Photonics	0000-515-11524
Shutter	Vincent Associates	LS6
Vibration Isolation Table	Minus k Technology	MK26
Specific reagents
Di-2-ANEPEQ (JPW 1114)	Life Technologies	D-6923	Voltage sensitive dye
Crym-EGFP Mouse Line	GENSAT (MMRRC)	STOCK Tg(Crym-EGFP)GF82Gsat/Mmcd	Sparsely expressing EGFP in Layer 5 cortical neurons