Fast Optical Recordings of Membrane Potential Changes From Dendrites of Pyramidal Neurons

Srdjan Antic,1,3 Guy Major,2,3 and Dejan Zecevic1,3

 1Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut 06520;  2University Laboratory of Physiology, Oxford University, Oxford, United Kingdom; and  3Marine Biological Laboratory, Woods Hole, Massachusetts 02543


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antic, Srdjan, Guy Major, and Dejan Zecevic. Fast Optical Recordings of Membrane Potential Changes From Dendrites of Pyramidal Neurons. J. Neurophysiol. 82: 1615-1621, 1999. Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment; 2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons; 6) the dye spread ~500 µm into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This will depend critically on how exactly are signals integrated by individual nerve cells functioning as complex operational units.

It is now well established that dendritic membranes of vertebrate CNS neurons contain active conductances such as voltage-activated Na+, Ca2+, and K+ channels (Magee et al. 1995; Segev and Rall 1997; Stuart and Sakmann 1994; Stuart et al. 1997). An important consequence of active dendrites is that regional electrical properties of branching neuronal processes will be 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 propagate from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. It is important to be able to perform these measurements in at least partially intact neuronal structures (isolated invertebrate ganglia or tissue slices of vertebrate CNS) to ensure the preservation of highly specific regional electrical properties of individual neurons and characteristic synaptic connections. This goal has not been fully accomplished in any neuron, vertebrate or invertebrate, due to technical limitations of experimental techniques that are presently available.

One way to achieve adequate spatial resolution is to use voltage-sensitive dye recording. Recently, the sensitivity of intracellular voltage-sensitive dye techniques for monitoring voltage transients from neuronal processes in situ, introduced by Grinvald et al. (1987), has been dramatically improved (by a factor of ~100) allowing direct recording of subthreshold and action potential signals from the neurites of invertebrate neurons (Antic and Zecevic 1995; Zecevic 1996). It is of considerable interest to apply this technique to dendrites of vertebrate CNS neurons in brain slices. In our initial effort (Kogan et al. 1995) we did not accomplish multisite recording of action potential signals due to a number of methodological difficulties. Most of these methodological problems are now resolved. Here we describe results showing simultaneous optical recording of electrical signals from multiple sites on apical, oblique, and basolateral dendrites of neocortical pyramidal neurons, and we discuss possibilities for further substantial improvements in the sensitivity of this approach.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Brain slice preparation and intracellular application of the dye

The 300-µm-thick coronal somatosensory neocortical slices from 13- to 25-day-old Wistar rats were prepared as described in Stuart et al. (1993). The solution used for slicing and for perfusion contained (in mM) 125 NaCl, 25 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2, pH 7.4 when bubbled with oxygen (95% O2-5% CO2). Somatic whole cell recordings were made with 7 MOmega patch pipettes using an Axoclamp 200B amplifier. The pipette solution contained (in mM) 115 potassium-gluconate, 20 KCl, 4 Mg-ATP, 10 phosphocreatine, 0.3 GTP, 10 HEPES (pH 7.3, adjusted with KOH), and 0.1-3 mg/ml JPW3028 (the di-methyl analogue of JPW1114; Antic and Zecevic 1995) voltage-sensitive dye. Glass pipettes were filled from the tip with dye-free solution by applying negative pressure for 3 min and were back-filled with dye solution. Intracellular staining was achieved by free diffusion of the dye from the pipette into the cell body. After allowing 40-60 min for diffusion, an outside-out patch was formed and the patch electrode removed. Antidromic or synaptically evoked action potentials were initiated in pyramidal neurons by extracellular bipolar stimulating electrodes placed in the white matter radially below the cell body. In some experiments the stained cell was repatched with standard (no dye) electrode and action potentials initiated by depolarizing the soma.

Optical recording

Optical recordings were done using an upright microscope (Leitz, Ortholux II) equipped with dark-field, bright-field, and fluorescence optics, two camera ports, and patch-pipette and bipolar stimulating electrode micromanipulators mounted on the stage. One camera port had the charge-coupled device (CCD) camera for visualizing neurons in slices under dark-field infrared illumination. The other camera port had a 464-element photodiode array that can be regarded as a very fast camera with low spatial resolution but very wide dynamic range (Centronic, Croydon Surrey, England). We used the optical recording system developed in Larry Cohen's laboratory at Yale University (NeuroPlex; OptImaging LLC; Fairfield, CT). Detailed descriptions of earlier versions have been published (see Wu and Cohen 1993). High-frequency noise in the recording was limited by 1-kHz cutoff frequency of a low-pass RC filter in the first stage amplifiers. An RC filter with cut-on frequency of 1.7 Hz was used to limit low-frequency noise. The output signals from the amplifiers were digitized using a data acquisition system (model DAP 3200e/214, MicroStar Laboratories, Bellevue, WA) in an IBM-PC computer. The system provides for 512 analogue inputs and a 769-kHz throughput rate with 12-bit A/D resolution. Recording was done in AC configuration; a 40-ms illumination period, to allow the amplifiers to settle to baseline, preceded the actual recording.

The brain slice was placed on the stage of a microscope and the fluorescent image of the stained cell projected by an objective onto the photodiode array positioned at the primary image plane. For recording optical signals we used a Zeiss 40X/0.75 NA or an Olympus 60X/0.9 NA objectives with the measured magnification, on our microscope, of ×53 and ×75, respectively. A 250W xenon arc lamp (Osram, XBO 250W OFR) powered by a low-noise power supply (Model 1700 × T; Opti-Quip, Highland Mills, NY) was used as a light source. Optical signals were recorded using an excitation interference filter of 520 ± 45 nm, a dichroic mirror with central wavelength of 570 nm and 610 nm barrier filter. A shutter was used to keep the light off between recordings; the preparation was illuminated for 70-84 ms for each trial. Data were displayed and analyzed with the NeuroPlex program (OptImaging, LLC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main objective of this work was to develop procedures that would allow the application of voltage-sensitive dye recording of electrical signals from multiple sites on neuronal processes of individual vertebrate neurons in brain slices (in situ). The experiments were carried out on layer V pyramidal neurons from 13- to 25-day-old rats at room temperature (23-25°C). A total of 23 cells was analyzed. The overall success rate in these experiments was ~50%. The limiting factor was the percent of successful giga-seal formations between the electrode and the cell membrane. The average somatic resting membrane potential and action potential amplitude for these cells at the beginning of the experiment were -53.6 ± 4.0 (SD) mV and 108.7 ± 5.3 mV, respectively. The duration of the action potential at half height was 1.67 ± 0.22 ms. The mean effective input resistance was 50.8 ± 8.0 MOmega . This value is higher than the result obtained by Stuart and Spruston (1998) at 37°, probably because our measurements were done at lower temperature.

Dye injection and spread

Individual pyramidal neurons in slices were observed using infrared dark-field video microscopy and selectively stained intracellularly with the voltage-sensitive dye by diffusion from a patch pipette. The major problem in injecting vertebrate neurons from patch pipettes was leakage of the dye from the electrode into the extracellular medium before the electrode is attached to the neuron. Patching requires pressure to be applied to the electrode during electrode positioning and micromanipulation through the tissue. This pressure ejects solution from the electrode. To avoid extracellular deposition of the dye that binds to the slice and produces large background fluorescence, the tip of the electrode was filled with dye-free solution, and the electrode was backfilled with dye solution. The amount of dye-free solution in the electrode tip and the applied pressure were empirically adjusted to ensure that no dye leaked from the electrode before the seal is formed. Usually, no pressure was applied before the electrode entered the slice. Low pressure (30 mBar) was used during electrode positioning in the slice, and the final pressure of ~100 mBar, necessary for cleaning the surface membrane of the cell, was applied immediately (1 s) before the seal formation. Using this method, we were able to routinely load neurons without increasing the background fluorescence of the surrounding tissue. An example of a stained neuron in a slice is shown in Fig. 1A. Also, Fig. 1 shows that brain slices (unlike some invertebrate ganglia) do not show any autofluorescence at the excitation/emission wavelengths and incident intensity we used.



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Fig. 1. Staining, pharmacological effects, and photodynamic damage in a neocortical pyramidal neuron. A: fluorescent image of a pyramidal neuron in the neocortical slice after 2 h incubation at room temperature and after the application of the fluorescent voltage-sensitive dye JPW2038. Excitation: 520 ± 45 nm; dichroic mirror: 570 nm, barrier filter: 610 nm. B: whole cell recordings of action potentials evoked by soma stimulation during staining and after a 135-min recovery period. The pharmacological effects of staining were fully reversible. C: photodynamic damage. Left: neuron with a patch-pipette attached to the soma; electrical recordings. Right: neuron without an attached electrode; optical recordings.

At the end of a 1-h diffusion period, the patch pipette was detached and the injected dye was allowed to spread for an additional 2 h before the start of optical recording. Within this time, dye would reach the apical tuft branches in layer I, ~1 mm from the soma (not shown). However, the concentration of the dye in these tuft dendrites and, thus the intensity of the fluorescent light, was too low to allow optical recording of action potential related signals. This limited the present recordings to distances <500 µm from the soma.

Pharmacological effects

Previous work has shown that certain voltage-sensitive dyes have little or no pharmacological effects when applied from the outside or inside to some invertebrate neurons within a limited concentration range (Antic and Zecevic 1995; Gupta et al. 1981; Ross et al. 1977).

We found that action potentials evoked in the soma of cortical pyramidal neurons were regularly affected, in a reversible way, during the staining period (Fig. 1B). The effect was strictly dye related, as determined in control experiments. Although the first spike in the evoked burst remained unaffected, the second action potential started to gradually change in amplitude ~40 min (30-50 min in different experiments) after the dye electrode was attached to the soma. Also, both the rising and falling phase of the second spike in a train were progressively prolonged. In two experiments, we found that reducing the dye concentration in the electrode substantially, to 0.1 mg/ml, did not noticeably reduce these effects. The correlation between the concentration of the dye in the electrode and observed pharmacological effects was not explored further.

The dye electrode was removed from the cell in Fig. 1B and the same neuron repatched with a standard patch pipette after a 2-h recovery period. Complete recovery of the evoked response was recorded in all of the neurons tested (n = 17).

Photodynamic damage

In our initial experiments on Helix neurons, we saw very little photodynamic damage. It was possible to average 100 trials with no noticeable change in action potential amplitude or waveform (Antic and Zecevic 1995). Brain slice preparations have generally been found to be more sensitive to photodynamic injury than invertebrate preparations (see Grinvald et al. 1982). We evaluated the photodynamic damage in cortical pyramidal neurons by determining the number of recording trials needed to induce noticeable changes in the size and shape of action potentials.

It was found that photodynamic damage was relatively small and tolerable if the patch pipette was not attached to the cell at the time of optical recording. In these measurements action potentials were evoked by an extracellular stimulating electrode positioned in the white matter. It was possible to acquire ~40 recording trials (illumination time: 84 ms/trial) before any effect of photodynamic damage was observed (n = 14; Fig. 1C; right panel).

However, the photodynamic damage was more pronounced in neurons that had an electrode attached to the soma. The left panel of Fig. 1C shows the typical, progressive photodynamic damage in a neuron exposed to high-intensity green light during five successive recording trials. The preparation was illuminated for 84 ms in each trial. In measurements from nine different cells, photodynamic damage limited optical recordings to three to five trials.

Multiple site optical recording

Figure 2 shows a typical, single trial multisite optical recording from a pyramidal neuron. The fluorescence changes were recorded by each of the 464 elements of the octagonal silicon diode array during the time the neuron was stimulated, by depolarizing the cell body, to produce the burst of two action potentials. Each trace in Fig. 2B represents the output of one photodiode for 44 ms. As evident from the figure, the optical signals were found in the regions of the array that correspond closely to the geometry of the cell (Fig. 2A).



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Fig. 2. Multisite optical recordings of action potential signals. A: outline of the 464-element photodiode array superimposed over the fluorescent image of the pyramidal neuron. B: single trial recording of action potential related optical signals. Each trace represents the output of 1 diode. Traces are arranged according to the disposition of the detectors in the array. Each trace represents 44 ms of recording. Each diode received light from a 14 × 14-µm square area in the object plane. Spikes were evoked by a somatic current pulse. C: comparison of electrical and optical recordings. Top panel: spatial average of optical signals from 8 individual diodes from the somatic region (· · ·) are superimposed on the electrical recording from the soma (---). Bottom panel: same electrical signal compared with a single-trial optical recording. D: action potentials from individual detectors at different locations along the basal, oblique and apical dendrites. Traces from different locations are scaled to the same height. The increasing delay between the signal from the somatic region and most proximal dendritic segments reflects the propagation velocity.

It is important to know how accurately voltage-sensitive dye signals represent electrical events. For many voltage-sensitive dyes, absorption and fluorescence changes are both fast and linear with membrane potential changes in the squid giant axon (Gupta et al. 1981; Salzberg et al. 1993). This was confirmed on pyramidal neurons. Figure 2C shows, on an expanded time scale, a comparison of the electrical recordings from the soma (smooth line) with the optical signals filtered to eliminate high-frequency noise (· · ·). The top panel in Fig. 2C is spatial average of single trial recordings from eight adjacent detectors that received light from the soma (region 2, Fig. 2, A and B), and the bottom panel is a single diode output from the same region. There is a good agreement between time courses of electrical and optical recordings.

Dye sensitivity (Delta F/F) and the signal-to-noise ratio

Optical signals associated with 110-mV action potentials, expressed as fractional changes in fluorescent light intensity (Delta F/F), were between 1 and 2% in recordings from neuronal processes.

With the sensitivity of the dye described and the level of noise in our recording system, relatively good signal-to-noise ratios could be obtained from regions on neuronal processes that are up to 500 µm away from the site of injection (soma). Figure 2D illustrates relatively good signal-to-noise ratio in a single trial recording from five different locations. The best signal-to-noise ratio was obtained from the proximal apical dendrite (S/N ~10). Recordings from regions on the apical dendrite 230 µm away from the soma had a signal-to-noise ratio of ~4.

Action potential propagation

Voltage-sensitive dye recording provides direct information about initiation and propagation of electrical signals. For example, it is straightforward to determine the direction and velocity of propagation of action potentials in neuronal processes as illustrated in Figs. 2D and 3. In the experiment shown in Fig. 2, the action potential burst was evoked by direct soma stimulation. In Fig. 2D, recordings from different locations, scaled to the same height, are compared on an expanded time scale. Each trace is a spatial average from two adjacent detectors. Both spikes in the burst originated in the proximity of the soma and propagated centrifugally along the apical and basolateral dendrites (action potential back-propagation) (Stuart and Sakmann 1994). The propagation velocity of the action potential shown in Fig. 2D was 0.22 m/s. The shape of the spikes changed with distance from the soma. At a distance of 230 µm from the soma, the half-width increased from 1.7 to 2.3 ms for the first spike and from 2.2 to 4.6 ms for the second spike in the burst. The average value for the propagation velocity of the action potential in the apical dendrite was 0.25 ± 0.06 m/s (n = 8).



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Fig. 3. Multisite monitoring of the propagation and broadening of the spike evoked by the stimulus delivered to the white matter. A larger area is covered by combining recordings from 2 measurements. A: fluorescence image of a pyramidal cell obtained by aligning charge-coupled device images taken at the 2 recording positions. B: multisite recording of the action potential signals obtained by synchronizing data from 2 measurements to the time of the stimulus (see text). Fluorescence recordings are divided by the resting light intensity to partially compensate for differences in signal size due to uneven staining and to differences in membrane area imaged onto individual detectors. C: action potential signals from individual diodes at different locations (1-5), scaled to the same height, are compared on expanded time base. D: color-coded representation of the data shown in B provides information about both spatial and temporal dimensions of the electrical event (see text).

The results shown in Fig. 3 illustrated multisite monitoring of the propagation and broadening of the spike evoked synaptically, by a stimulus delivered to the white matter. In this experiment we monitored a larger area of the injected neuron by combining recordings from two different measurements (low magnification objectives are not effective in recording from a larger area because they have significantly lower numerical apertures and thus low light gathering power). Two adjacent segments of the neuron were monitored, one at a time, and the temporal and spatial information was combined "off-line." The data files were synchronized to the time of the stimulus and aligned spatially according to CCD images taken at the two recording positions (Fig. 3A). Nine trials were averaged to improve the signal-to-noise ratio. Only a subset of detectors in the array were sampled to increase the frame rate to 2.5 kHz; detectors that were omitted appear flat in Fig. 3B. In Fig. 3C recordings from individual detectors from five different locations, scaled to the same height, are compared using an expanded time scale to determine the direction of spike propagation. Each trace is a spatial average from four adjacent detectors. The earliest spike was generated in the somatic region and back propagated peripherally into dendritic processes. This sequence of events is obvious in the color-coded representation of the data. Membrane potential changes in dendritic processes are shown at nine different times in Fig. 3D. The color scale is in relative units with the peak of the action potential shown in red. In this experiment the largest back-propagation delay (1.3 ms) was measured between the soma and the most distal (460 µm) region on the apical dendrite (location 5 in B) corresponding to a propagation velocity of 0.35 m/s. The largest propagation delay measured in the basolateral dendrites was 0.5 ms over a distance of 140 µm corresponding to velocity of 0.28 m/s.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These results demonstrate that multisite voltage-sensitive dye recording permits detection of temporal and spatial dynamics of action potential activity in the processes of vertebrate neurons in brain slices (in situ). An improvement in the sensitivity by an additional factor of 10 would be an important leap forward because that would allow the analysis of the integration of subthreshold signals in neuronal processes that precede action potential generation. We have evidence that there are no fundamental limitations that would prevent such an improvement (see Dye sensitivity (Delta F/F) and the signal-to-noise ratio).

Multiple site recording

The values for the velocity of spike propagation recorded optically in the apical dendrites are in the range of results obtained previously by electrical recordings (Buzsaki and Kandel 1998; Spruston et al. 1995; Stuart and Sakmann 1994; Stuart et al. 1997). Our measurements of the spike width (at half height) at different distances from the soma (Fig. 2D) gave values that are similar (Stuart and Sakmann 1994) or slightly smaller (Schiller et al. 1995) than previously reported. The measurements of action potential signals from the basal and oblique dendrites did not show any obvious evidence that their electrical properties are different from those determined in the apical dendrite of neocortical and hippocampal pyramidal neurons (Regehr et al. 1993; Stuart and Sakmann 1994).

Although the dye signal is proportional to the membrane potential change, it is not possible to determine the actual voltage change from these optical recordings. The proportionality between light intensity and membrane potential, after normalizing for differences in the resting light intensity, depends on the ratio of the amount of dye that is bound to membranes that do not change potential (inactive dye) to the amount of dye that is bound to the excitable membrane being monitored (active dye). It is generally the case that this ratio is unknown and different for different regions of a neuron. Thus calibration of all detectors cannot be done by calibrating the optical signal from any single site. However, in many measurements the absolute calibration of optical signals in terms of voltage is not critical. Many conclusions depend on the comparison of relative amplitudes of signals from the same location under different conditions and on timing information, which is directly obtained from voltage-sensitive dye recordings.

Possible improvements

INJECTION, SPREAD, AND PHARMACOLOGICAL EFFECTS OF THE DYE. The pharmacological effects of the dye introduced intracellularly were reversible and thus could be neglected. The reversible nature of these effects suggests that they might be the result of the high concentration of the lipophilic dye in the membranes close to the opening of patch electrode. Further spread of the dye into neuronal processes during 2 h of incubation period apparently equilibrates the concentration of the dye throughout the neuron to a tolerable level.

Multisite recording from neuronal processes was limited to distances <500 µm from the soma. The signal-to-noise ratio in these measurements (that are limited by dark noise) is dependent linearly on fluorescence intensity, which is a function of the amount of dye that reaches distal processes. It is therefore essential to optimize the staining protocol for recording from distal processes. An obvious improvement would be to extend the incubation period from 2 to 4-8 h. Other investigators showed that it is possible to maintain viable slices for >12 h (Chen et al. 1996; slices maintained for up to 4 days). Alternatively, it is possible to attach a patch electrode with the dye to a distal region on a dendrite and allow the dye to diffuse into terminal dendritic branches (see Markram and Sakmann 1994; Schiller et al. 1995). This approach will shorten the time needed for the spread of the dye.

Photodynamic damage

A surprising finding was that photodynamic damage in these measurements strongly depended on whether the patch electrode, in a whole cell configuration, was attached to the cell body. The damaging effect was much more pronounced in stained neurons with the patch electrode attached, independently of whether the electrode solution contained the dye or not. When the optical recording was done from stained neurons that did not have the patch electrode attached to the soma, photodynamic damage was dramatically reduced (Fig. 1C).

The precise mechanism of photodynamic damage is not known. Photolysis of some voltage-sensitive dyes is reported to produce both singlet oxygen and other reactive oxygen species, including oxygen radicals (Feix and Kalyanaraman 1991; Krieg 1992). It is possible that the wash out of some intracellular components (enzymes, antioxidants, and free radical scavengers) through the patch pipette might be responsible for the reduced ability of the cell to prevent harmful oxidative processes caused by high-intensity light.

The level of photodynamic damage in cells with no electrode attached already allows meaningful voltage-sensitive dye measurements from vertebrate neurons. If the presence of an electrode is necessary, sharp electrodes or the perforated patch technique could be used. Further reduction in photodynamic damage might be obtained by exploring the relationship between the phototoxic effects and light intensity. It is well established from the studies in radiotherapy and photodynamic therapy that toxic effects depend on dose-rate (Veenhuizen and Stewart 1995). Thus significant beneficial effects might be expected from determining the optimal compromise between incident light intensity, photodynamic damage, and signal-to-noise ratio.

Furthermore, it is reasonable to assume that the overall photodynamic damage would be reduced if the soma were selectively protected from excitation light by positioning a screen in the light path. Also, because photodynamic damage is dye dependent (Gupta et al. 1981; Ross et al. 1977), synthesis of additional analogues of JPW1114 may be useful. Finally, application of antioxidant agents and/or intermittent removal of oxygen from the medium during short recording periods might further reduce photodynamic damage.

Dye sensitivity (Delta F/F) and the signal-to-noise ratio

The dominant noise in recordings from neuronal processes with the photodiode array was dark noise (rather than optical shot noise), because the resting light levels were relatively low. The fluorescent photocurrents, detected by individual elements of the array from the typical snail neuron processes, with 250W xenon arc-lamp illumination and 10X, 0.4 NA objective, were ~1 nA (light intensity from the cell body was ~100 times higher).

Under these conditions improvements in the signal-to-noise ratio might be achieved using several approaches. 1) The most promising strategy is dark noise reduction. It is possible to significantly lower the dark noise in the recording system toward the theoretical minimum (optical shot noise) set by the statistical nature of photon emission and detection. The dark noise could be reduced by making the active area of individual photodetectors smaller and by cooling the photodiode-amplifier circuit. Both of these approaches are used in high-performance, low-noise CCD cameras with large dynamic range that have recently become available (FastOne, Pixel Vision, Beaverton, OR). In preliminary tests with this camera, we found that the signal-to-noise ratio at low light levels is larger by a factor of 10-15 compared with the photodiode array used in the experiments presented here. 2) Better dyes; we are currently testing analogues of the positively charged amino-naphthyl fluorescent dye JPW1114 for larger optical signals. Several additional molecules are in the process of being synthesized in the laboratory of Dr. Leslie Loew (University of Connecticut Health Center). 3) Higher light intensity; the signal-to-noise ratio, with the same dye, should increase linearly with increases in light intensity. It is possible to increase fluorescence intensity by using objectives with higher light-gathering power. This approach will have to be evaluated against possible negative effects of increased photodynamic damage. 4) Dye mobility; it would be useful to improve the mobility of voltage-sensitive dyes to ensure satisfactory staining of distant small processes within a reduced time for diffusion. One way to improve the mobility of amino-naphthyl dyes is to synthesize more hydrophilic analogues with the same chromophore. Another possible way to improve mobility is to use "carrier" molecules designed for the delivery of water insoluble compounds (beta-cyclodextrins; Sigma).

Spatial and temporal resolution

Spatial resolution in voltage-sensitive dye recording is determined by a number of factors. These include the magnification and the depth of field of the objective, the number of pixels of the photodetector, and light scattering properties of the preparation. With our recording system it was possible to distinguish signals from regions that are 13 µm apart (Fig. 2). Spatial resolution could be further improved by using an array of more elements. Also, higher magnification objectives can be used to image specific parts of neuronal arborizations in more detail.

Our temporal resolution was determined by the fastest acquisition rate of 0.6 ms per full frame with 464 pixels. This frame rate was not a limiting factor; faster sampling rates were achieved by restricting sampling to a subset of individual detectors under software control (Fig. 3).


    ACKNOWLEDGMENTS

We are grateful to L. Loew and J. Wuskel for kindly providing dyes and to L. Cohen and F. Helmchen for helpful comments on the manuscript.

This work was supported by National Science Foundation Grants IBN-9604356 and IBN-9812301.


    FOOTNOTES

Address for reprint requests: D. Zecevic, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 April 1999; accepted in final form 10 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society