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Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
1Correspondence: Physiology, 333 Cedar Street, New Haven, CT 06520, USA. E-mail: antic{at}fred.med
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INTRODUCTION |
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 TOP INTRODUCTION RESULTSFUTURE DIRECTIONSREFERENCES |
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In all of these experiments, the source of the optical signals are
voltage-sensitive dyes. These are dye molecules that bind to membranes
and change their spectral properties (absorption and/or fluorescence)
in response the changes in membrane potential (for reviews see refs
1
2
3
4
5
). The dyes that we have used are both linear and fast
(6
, 7)
; they respond to changes in membrane potential with
a time constant of <10 µs.
The first kind of measurement uses the optical signal of membrane potential to determine the spread of potential in the processes of an individual neuron. The second kind of measurement uses the dyes to monitor the spike activity in many individual cell bodies in an invertebrate ganglion while that ganglion is generating a behavior. The third kind of measurement records the population signals representing the average change in membrane potential from many cells and processes in a vertebrate central nervous system (CNS). All three kinds of recordings have provided information about the function of the nervous system that was previously unobtainable.
The measurements were made with a 464 element silicon photodiode array
system (24x24 grid with corner detectors omitted) with parallel
readout (NeuroPlex; RedShirt Imaging, LLC, Fairfield, Conn.) placed in
the image plane formed by a microscope objective. With this many
detectors it is easy to sample a complete frame every millisecond. The
parallel readout allows the measurement of a large number of photons
(up to 1010/ms), which yields a dynamic range of
up to 100 dB. (For additional details about the apparatus and
measurements see refs 8
, 9
).
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RESULTS |
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TOPINTRODUCTIONRESULTSFUTURE DIRECTIONSREFERENCES |
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11
12
).
An important consequence of active dendrites is that the electrical
properties of these processes will be 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, vertebrate, or invertebrate, because of
the technical limitations of experimental measurements that use
electrodes. To achieve better spatial resolution, it is necessary to
turn from direct electrical recording to indirect, optical measurements
using voltage-sensitive dyes. Recently, the sensitivity of
intracellular voltage-sensitive dye techniques for monitoring neuronal
processes in situ has been improved (by a factor of ~150),
allowing direct recording of subthreshold and action potential signals
from the neurites of invertebrate neurons (13
, 14)
. The
improvement in signal-to-noise ratio is based on previous experience
from other laboratories (15
, 16)
and on 1)
finding an intracellular dye that gives a relatively large fractional
change in fluorescence and 2) improvements in the apparatus
to increase the incident light intensity, lower the noise, and filter
more efficiently. Encouraging results have also been obtained in
preliminary studies on vertebrate CNS neurons in brain slices (17,
17a).
A typical result of multi-site optical recording from an individual
invertebrate nerve cell in situ is shown in Fig. 1A
. To understand the functional organization of this neuron, experiments
were carried out to determine the position of the action potential
trigger zones in different processes. The image of the cell, stained by
intracellular injection of the fluorescent voltage-sensitive dye
JPW1114, was projected by an objective onto the array of photodiodes as
indicated in the Fig. 1Aa
. This panel represents the
multi-site recording of action potential signals, evoked by a
transmembrane current step, from axonal branches Br2, Br3, and Br4.
Optical signals associated with action potentials, expressed as
fractional changes in fluorescent light intensity (
F/F), were ~1%
in recordings from the processes. With these measurements, it is
straightforward to determine the direction and velocity of action
potential propagation in the processes. The direction of propagation is
clear from the color-coded representation of the data (Fig. 1Ab
). This panel shows the potential changes in the
branching structure at nine different times separated by 1.6 ms. The
red color corresponds to the peak of the action potential. The panels
shows that the action potential trigger zone is located at position 2
and ortho- and antidromic spread of the nerve impulse occurs from this
site. The earliest spike was evoked ~1 mm away from the soma. The
spike initiation segment in the axon is ~300 µM in length. Under
normal conditions, slow depolarizing voltage pulses applied to the soma
are electronically spread into the processes with little attenuation
because of the very long axonal space constant for slow voltage
changes. These depolarizing pulses initiate action potentials at remote
sites in the processes that are characterized by higher excitability
than that of neighboring segments.
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Both the finite space constant for membrane potential changes in the processes (hundreds of microns for potential changes around the resting potential) and the blurring as a result of light scattering in the Helix ganglion limit the maximum useful spatial resolution in this kind of measurement. The 24 x 24 pixel resolution of NeuroPlex appears to be adequate in some circumstances.
Action potentials recorded from many individual neurons in an
Aplysia ganglion
Nervous systems are made up of large numbers of neurons, and many
of these are simultaneously active during the generation of behaviors.
The original motivation for developing optical methods was the hope
that they could be used to record all of the action potential activity
of all the neurons in simpler invertebrate ganglia (15)
.
Techniques that use microelectrodes to monitor activity are limited in
that they can observe single cell activity in only as many cells as one
can simultaneously place electrodes (typically two or three neurons).
Obtaining information about the activity of many cells is important for
understanding the roles of the individual neurons in generating
behavior and for understanding how nervous systems are organized.
In the first attempt to use voltage-sensitive dyes in ganglia
(18)
, we were fortunate to be able to monitor activity in
a single neuron because the photodynamic damage was severe and the
signal-to-noise ratio small. Now, however, with better dyes and
apparatus, the spike activity of hundreds of individual neurons can be
recorded simultaneously (19
, 20)
. In the experiment
described below, we measured the spike activity of ~50% of the
~1,000 cells in the Aplysia abdominal ganglion.
Opisthobranch molluscs have been a preparation of choice for this kind
of measurement because their CNSs have relatively few, relatively large
neurons, and almost all of the cell bodies are fully invaded by the
action potential.
Because the image of a ganglion is formed on a rectilinear diode array,
there is no simple correspondence between images of cells and
photodetectors. Thus, a sorting step is required to determine the
activity in neurons from the spike signals on individual photodiodes.
Although this sorting step is tedious, difficult, and almost certainly
results in errors, different individuals from the same laboratory and
from different laboratories have obtained similar results. The result
from one data set is shown in the raster diagram of Fig. 1B
.
The start of the 0.5 s mechanical stimulus occurred at the time
indicated by the ‘Stim’ at the bottom. The bottom trace shows the
gill movement. There were 110 neurons whose activity was detected
optically. Similar results have been obtained by Nakashima et al.
(20)
. Because the signal-to-noise ratio in the
measurements was not large, it seemed likely that this recording was
not complete. Wu et al. (21)
estimated that ~50% of the
active neurons were detected. Thus, the actual number of activated
neurons during the gill-withdrawal reflex was ~250.
We were surprised at the large number of neurons that were activated by
the light touch. Furthermore, a substantial number of the remaining
neurons are likely to be either inhibited by the stimulus or receive a
large subthreshold excitatory input. It is almost as if the
Aplysia nervous system is designed such that every cell in
the ganglion cares about this (and perhaps every) sensory stimulus. In
addition, >1000 neurons in other ganglia are activated by this touch
(22)
. Clearly, information about this very mild and
localized stimulus is propagated widely in the Aplysia
nervous system. These results force a more pessimistic view of the
present understanding of the neuronal basis of apparently simple
behaviors in relatively simple nervous systems. Elsewhere we present
arguments suggesting that the abdominal ganglion may function as a
distributed system (21
, 22)
.
The fact that we can monitor the activity of a large fraction of the neurons during the behavior means that we can attempt to relate the activity of individual (groups) of neurons to the behavior.
In this kind of measurement the optimal signal-to-noise ratio will be obtained when the size of the image of an individual neuron and the size of a photodetector are identical. A smaller signal-to-noise ratio will result either if the light from a single cell body falls on many detectors or if the light from many neurons falls on a single detector. Thus, the 464 element array was approximately ideal for the Aplysia preparation containing ~1,000 neurons.
Population signals from the turtle olfactory bulb
In a measurement from a vertebrate brain stained by
superfusing a solution of the dye over the surface, each photodetector
will receive light from a large number of neurons and neuronal
processes. Because of the blurring resulting from light scattering and
from signals from layers that are out-of-focus, the X-Y spatial
resolution from a measurement of an intact brain structure using
ordinary microscopy cannot be better than 100–200 µM
(23)
. This limitation means that a 24 x 24 array is
adequate for brain areas up to ~4 x 4 mm (which would include
the entire olfactory bulb in the turtle). In the measurements described
below, each detector received light from a 150 µM square area (a
volume of ~0.1 mm3) of bulb, which contains
thousands of neurons and processes. The voltage-sensitive dye signal
will be a population average of the change in membrane potential in all
of these membranes. The signals will represent the coherent
(synchronous) activity of the neurons in the areas imaged onto the
individual photo-diodes.
E. D. Adrian first recorded spontaneous oscillations in field
potential recordings from the olfactory bulbs of the cat, rabbit, and
hedgehog and showed that odors would increase their magnitude and
frequency (23a)
. The induced oscillations were the
same for different odors (23a
, 24
).
The spatial distribution of the field potential oscillations was
studied in the rabbit by Freeman (24)
and Freeman and Di
Prisco (25)
using an array of 64 implanted electrodes.
They found that "the same time series held for almost all channels on
almost every burst." The largest phase difference between distant
sites on the bulb was a small fraction (5–8%) of the cycle period,
implying that the signals would mainly appear over the entire bulb as
standing waves of depolarization.
We made voltage-sensitive dye recordings of the response to odors in
the olfactory bulb of the turtle. Preliminary evidence
(26a)
suggests that these optical measurements
have a spatial resolution ~25 times better than local field potential
measurements. Our results in the turtle are quite different from those
found by Freeman and Di Prisco (25)
. We found that odor
application to the nose elicited three different oscillations in the
turtle bulb. These oscillations differed in location, frequency,
latency, and concentration dependence. Figure 1C
illustrates
the outputs of five photodiodes (1–5) positioned in a rostral to
caudal order. There is a short latency DC signal (arrow) seen on all
five detectors. The next signal is an oscillation (~14 Hz) labeled
medial, which is seen best isolated at position 3. This is followed by
the rostral signal, which is seen best isolated at position 1. The
rostral has the highest frequency (16 Hz). The caudal signal has the
longest latency and lowest frequency (a frequency that is very close to
one half of the rostral frequency). This signal is best isolated at
position 5. Detectors 2 and 4 come from regions where signals overlap
(rostral and medial for detector 2 and medial and caudal for detector
4). The medial signal occurred at the lowest odorant concentration;
both the rostral and the caudal signals could often not be detected at
concentrations where the medial signal was relatively large. The
rostral and caudal signals appeared to propagate. The rostral signal
began in the most rostral part of the bulb and propagated caudally. The
caudal signals had less regular propagation patterns, but they too were
not simple standing waves.
The difference between our results in the turtle and the earlier
results of Freeman (24)
in the rabbit may result from a
species difference. However, another possible explanation for the
difference is that our voltage-sensitive dye measurements have a
spatial resolution that would facilitate the detection of spatially
separate patterns. Additional experiments will be required to
distinguish these two possibilities.
The fact that these odor induced oscillations involve coherent activity of a large number of neurons suggests that they might be functionally important. However, their functional meaning and/or importance are not known. Future experiments will be directed at determining whether these signals are different for different odors and thereby have some role in odor recognition. Another possibility is that the coherent activity is related to the animal’s arousal state.
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FUTURE DIRECTIONS |
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, 28)
. An identified cell class (motoneurons) was selectively
stained. Spike signals from individual neurons were measured in lamprey
experiments (29)
. Second, is the use of cell-type specific
staining developed for fluorescein (30)
. It might be
possible to use a similar strategy to selectively stain cells with
voltage-sensitive or ion-sensitive dyes. Third, Siegel and Isacoff
(31)
constructed a genetically encoded combination of a
potassium channel and green fluorescent protein. When introduced into a
frog-oocyte, this molecule had a (relatively slow) voltage dependent
signal with a fractional fluorescence change of 5%. Optical recording already provides unique insights into brain activity and organization. Improvements in sensitivity or selectivity would make these methods even more powerful.
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ACKNOWLEDGMENTS |
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
