Center for Molecular and Behavioral Neuroscience, Rutgers, The State University of New Jersey, Newark, New Jersey 07102
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ABSTRACT |
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Henze, Darrell A., Zsolt Borhegyi, Jozsef Csicsvari, Akira Mamiya, Kenneth D. Harris, and György Buzsáki. Intracellular Features Predicted by Extracellular Recordings in the Hippocampus In Vivo. J. Neurophysiol. 84: 390-400, 2000. Multichannel tetrode array recording in awake behaving animals provides a powerful method to record the activity of large numbers of neurons. The power of this method could be extended if further information concerning the intracellular state of the neurons could be extracted from the extracellularly recorded signals. Toward this end, we have simultaneously recorded intracellular and extracellular signals from hippocampal CA1 pyramidal cells and interneurons in the anesthetized rat. We found that several intracellular parameters can be deduced from extracellular spike waveforms. The width of the intracellular action potential is defined precisely by distinct points on the extracellular spike. Amplitude changes of the intracellular action potential are reflected by changes in the amplitude of the initial negative phase of the extracellular spike, and these amplitude changes are dependent on the state of the network. In addition, intracellular recordings from dendrites with simultaneous extracellular recordings from the soma indicate that, on average, action potentials are initiated in the perisomatic region and propagate to the dendrites at 1.68 m/s. Finally we determined that a tetrode in hippocampal area CA1 theoretically should be able to record electrical signals from ~1,000 neurons. Of these, 60-100 neurons should generate spikes of sufficient amplitude to be detectable from the noise and to allow for their separation using current spatial clustering methods. This theoretical maximum is in contrast to the approximately six units that are usually detected per tetrode. From this, we conclude that a large percentage of hippocampal CA1 pyramidal cells are silent in any given behavioral condition.
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INTRODUCTION |
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The study of how the activity of
single neurons contribute to network computations requires the ability
to record the simultaneous activity of large numbers of neurons. The
use of multichannel extracellular recording techniques has improved
single-unit sorting by taking advantage of the temporal coherence of
spikes from closely-spaced recording sites (Buzsaki and Kandel
1998; Drake et al. 1988
; McNaughton et
al. 1983
; Recce and O'Keefe 1989
; Wilson
and McNaughton 1993
). An important outstanding issue is what is
the maximum number of simultaneously recordable neurons that can be
achieved using extracellular recording techniques. The maximum number
of detectable cells is expected to vary across brain regions depending
on variables such as neuronal density and activity. Here we focus our
attention on unit detection in the rat hippocampal area CA1.
Extracellular recording methods traditionally provide information only on whether a neuron fires a spike or not. To obtain information about subthreshold variations in neuronal membrane potential, it is necessary to use intracellular recording methods. In addition, intracellular recording has the added advantage that it allows subsequent morphological identification of the recorded neuron. However, intracellular recordings typically are obtained from one cell at a time under nonphysiological conditions, e.g., in tissue slices and anesthetized brains. Since the extracellularly recorded spikes arise due to transmembrane currents and concurrent changes in intracellular membrane potential, it should be possible to obtain more detailed information about intracellular changes from close examination of the extracellular spikes. However, this requires a better understanding of the relationship between an intracellular action potential and the extracellular spike.
The present experiments provide important information about the sensitivity of the tetrode recording method and investigate the relationship between intracellular action potentials and extracellular spikes. Our investigations ask the following questions: what is the precise temporal relationship between the intracellularly recorded somatic and dendritic action potential and its extracellularly recorded counterpart? Does variation in the intracellular action potential contribute to amplitude and shape variability of extracellular spikes? How close does a neuron have to be to a tetrode to be detected and subsequently isolated during analysis?
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METHODS |
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Simultaneous intracellular-extracellular recording in anesthetized rats
Thirty rats (350-450 g) of the Sprague-Dawley strain (Hilltop
Labs, Scottsdale, PA) were anesthetized with urethan (1.5 g/kg; Sigma)
and placed in a stereotaxic apparatus. The body temperature of the rat
was kept constant by a small animal thermoregulation device. The scalp
was removed, and a small (1.2 × 1.2 mm) bone window was drilled
above the hippocampus (centered at AP = 3.5 and L = 2.5 mm
from bregma) for extra- and intracellular recordings. A pair of
stimulating electrodes (100 µm each, with 0.5-mm tip separation) were
inserted into the left fimbria-fornix (AP =
1.3, L = 1.0, V = 3.95) to stimulate the commissural inputs. Extracellular and
intracellular electrodes were mounted on two separate manipulators on
opposite sides of a Kopf stereotaxic apparatus. The horizontal axes of
the two manipulators were parallel. The manipulator of the
extracellular electrode was mounted at a 10° angle from vertical to
permit the subsequent placement of the intracellular electrode. The
optimal distance between the electrodes at the brain surface to cause
the tips to arrive at the same point at the level of the hippocampus (2 mm deep) was calculated to be ~370 µm. The extracellular electrode
was lowered into the cell body layer of CA1 by monitoring for the
presence of unit activity and evoked field potentials. Once the
intracellular and extracellular electrode tips were placed in the
brain, the bone window was covered by a mixture of paraffin (50%) and
paraffin oil (50%) to prevent drying of the brain and decrease
pulsation. The intracellular micropipette was then advanced into the
region near the extracellular electrode, and an intracellular recording
from a CA1 pyramidal cell was obtained. If no extracellular and
intracellular pairs were encountered after advancing the micropipette
through the CA1 pyramidal layer and stratum radiatum, the intracellular
electrode was withdrawn, and a new intracellular electrode track was
made from the cortical surface.
Detection of intracellular action potentials and extracellular spikes
Intracellular action potentials were detected by examining the first derivative of the membrane potential for peaks. Once a peak was detected in the first derivative, the actual peak of the AP was determined from the point where the first derivative crossed back through zero. The extracellular spikes were extracted based on the occurrence of the intracellular action potential.
The extracellular "tetrodes" (Recce and O'Keefe
1989) consisted four 13-µm polyimide-coated nichrome wires
(H. P. Reid, Palm Coast, FL) bound together by twisting them and
then melting their insulation (Gray et al. 1995
). In
five rats, the extracellular electrode consisted of a single 60-µm
wire. The extracellular signal was amplified 1,000-8,000 times using a
DC amplifier and filtered at 1-3,000 Hz (Model-12, Grass Instruments,
West Warwick, RI). The micropipettes for intracellular recordings were
pulled from 2.0-mm capillary glass (World Precision Instruments,
Sarasota, FL) and filled with 1 M potassium acetate (Fisher Scientific, Pittsburgh, PA). The electrode solution also contained 2% biocytin (Sigma, St. Louis, MO) for single-cell labeling of all recorded cells.
In vivo electrode impedances were between 60 and 110 M
as measured
by the bridge balance circuit of the amplifier (Axoclamp-2A, Axon
Instruments, Foster City, CA). Once stable intracellular recordings
were obtained (resting membrane potentials less than
55 mV), evoked
and passive physiological properties of the cell were determined. All
electrophysiological signals were digitized [sampled at 10, 20, 25, or
50 kHz at 12 or 16 bit resolution (R. C. Electronics, Santa
Barbara, CA)] and stored on computer disk for later analysis.
Histological procedures
After the completion of the intracellular physiological data collection, biocytin was injected through a bridge circuit using 500-ms depolarizing pulses at 0.6-2 nA at 1 Hz for 10-60 min. Neuronal activity was followed throughout the procedure, and the current was reduced if the electrode was blocked and/or the condition of the neuron deteriorated. Two to 12 h after the injection, the animals were given an urethan overdose and then perfused intracardially with 100 ml physiological saline followed by 400 ml of 4% paraformaldehyde and 0.2% glutaraldehyde dissolved in phosphate buffered saline (pH = 7.2). The brains were then removed and stored in the fixative solution overnight. Sixty-micrometer-thick coronal sections were cut and processed for biocytin labeling. The histological sections were also used to verify the position of the extracellular recording electrodes and the track made by the recording pipette. The orientation between the labeled neuron and the tip of the tetrode was reconstructed by tracing the neuron soma and the tetrode track using a drawing tube. The distance between the neuron and the electrode tip was measured from the traced image. If the tetrode tip was in a different section than the neuronal soma, the Pythagorean theorem was used to calculate the distance.
Extracellular recording in behaving animals
SURGERY AND RECORDING.
The surgical procedures, electrode preparation, and implantation
methods have been described in a previous study (Csicsvari et
al. 1999a). The data from that previous study was re-examined for the analyses presented here. In short, 19 male rats were implanted with either wire "tetrodes" or silicon electrode arrays that were used for recording of neuronal activity. Silicon electrode arrays were
fabricated using integrated circuit technology. The shanks of the
silicon probes were separated by either 150 or 300 µm. Each shank
contained four or six recording sites (9 × 12 µm platinum pads)
with 25-µm vertical spacings (Ylinen et al. 1995
). The
electrodes were attached to a multidrive array, and the electrodes were
slowly advanced until they reached the CA1 pyramidal layer. Two 50-µm single tungsten wires (with 2 mm of the insulation removed) were inserted into the cerebellum and served as ground and reference electrodes.
DATA PROCESSING.
Electrical activity was recorded during sleep while the rat was in its
home cage (Csicsvari et al. 1999a). After amplification (5,000-10,000 times) and band-pass filtering (1 Hz to 5 kHz; Model 12-64 channels; Grass Instruments), field potentials and extracellular spikes were recorded continuously and digitized (10-50 kHz at 12 or 16 bit resolution, R. C. Electronics) and stored on computer disk for
later analysis. Recording sessions lasted from 15 to 50 min. The
separation and analysis of the extracellular units was accomplished
using standard cluster based separation methods detailed elsewhere
(Csicsvari et al. 1999a
).
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RESULTS |
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Simultaneous extracellular and intracellular recordings were
obtained from area CA1 of the dorsal hippocampus of anesthetized rats.
In cases where the electrode placements were accurate, simultaneous spikes were observed in the extracellular and intracellular recordings as the intracellular electrode was advanced through the tissue and
attempts were made to obtain stable intracellular recordings. As many
as six or seven "paired" extracellular and intracellular signals
were seen for a single track as the intracellular electrode impaled
either dendrites or somata as it was advanced through the s. oriens, s.
pyramidale, and s. radiatum (~90% of all intracellular electrode
tracks yielded some simultaneous activity on the intracellular and
extracellular electrodes). Of the numerous attempts to obtain stable
intracellular recordings from cells also recorded by the tetrode, we
were able to obtain recordings from 33 neurons. With practice, >75%
of intracellular electrode tracks passed through the recordable volume
of the extracellular tetrode. A subset of 22 neurons were selected for
further analysis because they had stable intracellular recordings with
resting potentials more negative than 55 mV as well as satisfactory
anatomical labeling. This subset consisted of 21 pyramidal cells and
one interneuron, all of which were morphologically identified by
staining for the intracellularly injected biocytin. The track of the
extracellular tetrode was reconstructed from the extracellular blood
cells present after the tetrode was withdrawn. Figure
1, A and B,
illustrates the arrangement of the recording electrodes with respect to
a single pyramidal cell as well as simultaneously recorded average
intracellular action potentials and extracellular spikes for that cell.
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Temporal relationship between extracellularly and intracellularly recorded action potentials
Figure 1C illustrates the main features of an average
intracellularly recorded action potential and the average
extracellularly recorded "spike" for the same cell illustrated in
A and B. The intracellular action potential had a
waveform typical of a recording obtained from the soma of CA1 pyramidal
cells (Kamondi et al. 1998). The wideband filtered (1 Hz
to 3 kHz) extracellular spike waveform was also typical for normal
tetrode recordings consisting of an initial large negative deflection,
followed by a wider and smaller positivity (Csicsvari et al.
1999a
). In some cases, the average extracellular spike was
preceded by a slow negativity that most likely reflected the field
electroencephalographic (EEG) signal corresponding to the intracellular
excitatory postsynaptic potential (EPSP) leading to the action
potential. The early large negative deflection of the extracellularly
recorded spike corresponded in time to the depolarizing phase of the
action potential. The later positive deflection of the extracellular
spike corresponded to the depolarizing and afterhyperpolarization
phases of the intracellular action potential.
We quantified the temporal relationship between the extracellular and
intracellular waveforms for three "fixed" time points: the start
time, the "peak" time, and the "end" time (the return to
threshold potential for intracellular, and the peak of the late
positivity for the extracellular). For the 11 somatic intracellular recordings obtained for this study, the average extracellular spike
started 0.01 ± 0.02 (SD) ms and ended 0.02 ± 0.14 ms
earlier than the corresponding points on the average intracellular
action potential. The action potential duration was calculated to be 1.28 ± 0.18 ms and 1.27 ± 0.19 ms from the intracellular
and extracellular waveforms, respectively. As expected, there was a
linear relationship between the intracellular and extracellular spike
durations across individual cells (R = 0.66, P < 0.03). Thus the duration of the intracellular
action potential (from onset to return to baseline; r +
d in Fig.
1C) is well approximated by the time elapsed between the
onset and the late positive peak of the extracellularly recorded waveform. The negative peak of the extracellular spike preceded the
peak of the intracellular somatic action potential by 0.20 ± 0.08 ms (ordinate values for black squares in Fig. 3, B and C).
Other relationships between the extracellular and intracellular
waveforms were also observed. In some cases, following the extracellular negative peak, the ascending component of the spike exhibited an initial fast slope followed by a period of a slower slope
(e.g., Fig. 4B, arrow) (see also Csicsvari et al.
1999a). This "notch" on the ascending phase corresponded in
time to the peak of the intracellularly recorded action potential.
Following the positive peak, the extracellular spike gradually returned to the baseline. Often the late positive peak was followed by a clearly
recognizable change in the slope (Figs. 1C and
2B,
), corresponding to the peak of the intracellular
spike afterhyperpolarization.
It has been proposed that the extracellular spike should be shaped like
the first derivative (with respect to time) of the intracellular
voltage (Brooks and Eccles 1947; Fatt
1957
; Freygang and Frank 1959
; Terzuolo
and Araki 1961
; but see Rall and Shepherd 1968
).
However, we have observed consistent departures from this simple model.
Whereas the waveforms were similar during the ascending phase of the
action potential (
r), they differed
substantially during the descending phase and spike
afterhyperpolarization (
d; Fig.
1C). We consistently observed that after the initial
ascending phase (
r), the intracellular
derivative followed a much faster trajectory than the extracellular spike.
It is important to note that the temporal relationships described above are for a wideband filtered (1 Hz to 3 kHz) extracellular waveform. Extracellular waveforms that have been filtered for higher frequencies will exhibit different relationships that will be dependent on the filter properties. Therefore we report only the relationship between the intracellular waveform and the wideband extracellular waveforms to permit comparison of data across laboratories that may use very different filters.
One additional cell was not included in the group analysis described in the preceding text. This cell was excluded based on the observation that it had an atypical extracellular waveform consisting of a large initial positivity. In this case, the tetrode appeared to have been closest to the cell body of all cells investigated. In fact, the exact relationship between the neuron and tetrode could not be determined because the red blood cell clot at the end of the extracellular tetrode track largely obscured the soma of the biocytin filled pyramidal cell. In an additional five cases, simultaneous intracellular and extracellular recordings of action potentials were achieved with a 60-µm single wire. The qualitative findings were the same as described for tetrode recordings.
Figure 2 illustrates the relationship between the extracellular and intracellular waveforms for a basket interneuron. Although the shape of the action potential is different from pyramidal cells, the qualitative relationship between the intracellular and extracellular waveforms was the same as described in the preceding text.
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Nine other pyramidal cell recordings were obtained with dendritic
intracellular impalements. The recordings were determined to be from
the dendrites based on the lack of a fast afterhyperpolarization and/or
an action potential peak value <0 mV (Kamondi et al.
1998). The simultaneously recorded extracellular signals
reflected the occurrence of the somatic action potential since the
tetrode was always placed in the pyramidal layer. Figure
3A shows an example where
sequential recordings were obtained from two cells. The first recording
was a somatic impalement that was later followed by a dendritic
impalement of a different neuron 240 µm deeper. For the dendritic
recording, the peak of the extracellular spike occurred 0.55 ms before
the intracellular peak, whereas for the somatic recording, the
extracellular spike occurred 0.28 ms before the intracellular peak. We
have previously reported that the amplitude of the intracellularly
recorded action potential varies directly as a function distance from
the soma (Kamondi et al. 1998
). Figure 3B
shows that the latency between the extracellular peak and intracellular peak for all 21 pyramidal cells also varies as a function of
intracellular action potential amplitude. If we assume that the
distance between the intracellular electrode tip and the soma is the
main reason for the variation in the average intracellular action
potential amplitude, then we can use the relationship we have
previously reported (4.9 µm/mV) (Kamondi et al. 1998
)
to estimate the relationship between extracellular-intracellular peak
latency and intracellular recording distance from the soma (Fig.
3C). These data support the conclusion that under our
conditions, the average action potential is initiated at the soma and
back-propagates into the dendrites. The average conduction velocity of
the back-propagating spike calculated from these data are 1.61 m/s.
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Correlated variations in amplitude and shape between intracellular and extracellular spikes
We also investigated whether there is a correlation between changes in intracellular and extracellular spike amplitude and shape. We examined the relationship between the intracellular and extracellular signals during bursts of action potentials evoked by intracellular current injection (Fig. 4). Intracellular depolarizing current steps induced an accommodating series of action potentials. Figure 4, A-C, shows the relationships during slow evoked burst activity (1st interspike interval ~10 ms). As expected for current-induced bursts, the intracellular waveform of the first action potential differed from subsequent action potentials. Over the course of the evoked depolarization, the amplitude of the action potential became smaller, and the slope of both the rising and falling phases decreased (lower traces in Fig. 4, B and C). These data also indicate that the amplitude of the somatic intracellular action potential changes due to a depolarization of the baseline membrane potential and not due to changes in the peak potential of the action potential. The observed intracellular waveform changes were accompanied by commensurate alterations of the extracellular waveforms. The amplitude of the extracellular spike decreased, the slope of the positive-going phase of the spike decreased and the "notch" became less prominent (Fig. 4, B and C). Figure 4D shows, for a different cell, the relationship between the extracellular spike amplitude and intracellular action potential amplitude for bursts of spikes occurring at a higher frequency (~5-ms inter-spike intervals). The best fit line (R = 0.40; P < 0.0001) illustrates the positive relationship between the intracellular and extracellular amplitudes. Taken together, these findings indicate that relative changes in the extracellular spike amplitude over the course of a recording session can be used to make predictions about relative changes in intracellular membrane potential at the time of the spike.
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Behavioral state effects on extracellular spike waveform
As shown in the preceding text, depolarization exerts an effect on
the amplitude and shape of the extracellular spikes (Fig. 4). We
examined whether the amplitude of the extracellularly recorded spikes
was affected by the state-dependent modulation of the membrane potential in nonanesthetized behaving animals. Extracellular spike amplitudes for individual units during rapid-eye movement (REM) sleep
associated with theta oscillation were compared with spikes from the
same units collected during slow wave sleep in the absence of theta
(Fig. 5). The amplitude of individual
spikes was measured from the negative to the positive peak of each
waveform. Spikes during sharp wave (SPW) bursts were omitted from the
analysis to avoid potential contamination from temporally overlapping
spikes during population bursts associated with sharp waves
(Buzsaki et al. 1983; Harris et al.
2000
). This comparison revealed that the mean spike amplitude
of individual pyramidal units and pyramidal layer interneuronal units
was lower during theta oscillations (P < 0.003; paired
Student's t-test). Based on the findings from Fig. 4, one
possible explanation for the relatively smaller spike amplitudes during
theta (Buzsaki et al. 1983
; Csicsvari et al. 1999a
) is that many of the action potentials of both pyramidal cells and pyramidal layer interneurons are initiated from more depolarized membrane potentials during theta activity. This is further
supported by the fact that the frequency of discharge of both pyramidal
cells and interneurons is higher during theta activity than during
"no-theta and no-sharp wave" periods.
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Field measurements of action potentials
The simultaneous intracellular and extracellular recordings from
single neurons also allowed us to examine the spread of spikes in the
extracellular space. We found that the amplitude of the average
extracellular spike decreased rapidly as a function of distance of the
tetrode tip from the soma. Figure
6A shows the amplitude of the
average extracellular spike versus the distance measured between the
end of the tetrode track and the intracellularly labeled cell. The
largest amplitude spikes were recorded <50 µm from the soma. At
distances >50 µm, individual small amplitude spikes (<60 µV)
still could be recognized in the extracellular recordings at the times
of the intracellular action potentials. However, the reliability of
unit separation for these low-amplitude units is decreased
significantly compared with spikes that are 60 µV. Under the
conditions used in our laboratory, most units that can be successfully
clustered are >60 µV (e.g., see lower limit of Fig. 5)
(Harris et al. 2000). At extracellular distances >140
µm from the soma (Fig. 6C), no extracellular spike
activity could be distinguished from the background noise activity even after averaging based on the occurrence of the intracellular action potentials. Unfortunately although we regularly observed extracellular spikes that were >250 µV in amplitude, we failed to obtain stable intracellular recording from such cells. We believe that this is due to
the technical challenge of obtaining recordings from a very specific
and small group of neurons that are very close to the tetrode tip (see
Fig. 6D).
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We have estimated the theoretical maximum number of simultaneously
recordable neurons in CA1 by a single tetrode based on our measurements
and an estimate of CA1 cell density of
300,000/mm3 (Aika et al. 1994).
According to our measurements (Fig. 6A), the amplitude of
the spikes <50 µm from the tetrode is
60 µV and is therefore
detectable and clusterable. Assuming a CA1 pyramidal layer thickness of
60 µm and a radius of 50 µm for effective cluster separation of
units, a single tetrode can record >60 µV amplitude spikes from
cells in a cylinder with a volume of 4.71 × 10
4
mm3. This volume is calculated to contain 141 cells. In reality, the number of cells in this volume probably is
smaller because the insertion of a tetrode (radius = 15 µm) will
damage or replace
13 neurons. Finally because all cells that can be
recorded from are
140 µm from the tetrode, theoretically one could
record from as many as 1,108 neurons with a single tetrode with perfect
recording and unit isolation methods.
Multiple recording of extracellular spikes along the somato-dendritic axis
In all of the experiments described in the preceding text, the
wire tetrode was placed in the pyramidal layer and therefore the
distances measured are mainly in the "lateral" direction at the
level of the pyramidal cell somata. By using double-shanked silicon
probes with six linearly placed recordings sites per shank (25-µm
intersite distances, 150-µm intershank distance), we were also able
to investigate the vertical spread of extracellular spikes
perpendicular to the pyramidal layer. Figure
7 shows such a recording. The
extracellular waveforms were averaged based on the occurrence of the
simultaneously recorded intracellular action potentials. The
extracellular channel with the largest amplitude spike was regarded as
the putative location of the soma in the dorsovental axis. Depending on
the relative position of the silicon probe, extracellular units could
be recorded from 100 µm above and below the putative location of
the soma (Fig. 7). In other recordings from behaving animals using
silicon tetrodes with 50-µm vertical separation, spikes were often
simultaneously present at all four recordings sites (data not shown).
Therefore we conclude that spike activity can be monitored at least
over a 200-µm vertical span.
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Coincident with the extracellular spike amplitude decrease, the rise
and decay slopes of the extracellular spike also decreased with
distance from the soma (Fig. 7). These changes in spike shape paralleled the action potential changes recorded in dendrites at
various distances from the soma (Kamondi et al. 1998),
although quantitative comparisons from simultaneously recorded
intradendritic and extradendritic measurement are not available. The
propagation velocity of action potentials, estimated from the distance
of the recording sites and the delay between the negative peaks of the
extracellular spikes, varied from 0.4 to 2 m/s in pyramidal cells
(n = 25) and from 0.5 to 1.5 m/s in pyramidal layer
interneurons (n = 6). These numbers agree with the
value calculated (1.61 m/s) from the simultaneous intracellular and
extracellular recordings presented in the preceding text.
A second feature of the multishank silicon probe electrodes is that
they have a fixed horizontal spacing (150 or 300 µm between the
shanks). Using these electrodes, we found only two cases in our
database from behaving animals (<0.5% of all clustered units) (Csicsvari et al. 1999a) when the extracellular spike,
recorded at one shank, was also recorded by the neighboring shank (Fig. 8). The co-occurrence of the same spike
was indicated by the cross-correlogram between units clustered
separately for each shank. In both cases, a large peak was present in
the cross-correlogram at 0 ms, surrounded by suppressed probability of
spikes within 5 ms. In other words, the cross-correlogram of these
independently clustered units resembled their individual
autocorrelograms. Both units were classified as putative interneurons,
located in s. oriens and in the pyramidal layer. Because similar
results were never observed for units that were judged to be pyramidal
cells, this suggests that the action potential-associated extracellular
current spread for pyramidal cells is substantially larger in the
vertical than in the horizontal direction. This is to be expected for
pyramidal neurons which have an open field (Hubbard et al.
1969
). Furthermore we assume that our success of recording some
interneurons with neighboring shanks can be explained by the oblique or
horizontal main dendrites these neurons possess (Freund and
Buzsaki 1996
).
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DISCUSSION |
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The main findings of this study are that the extracellularly recorded spike reveals information about the shape of the intracellular action potential and membrane polarization and that a single tetrode placed in the CA1 pyramidal layer should theoretically be able to simultaneously record from ~100 neurons.
Relationship between intracellular action potentials and extracellular spikes
Investigating the rules that underlie neural representation requires simultaneous monitoring of a large number of cells in a behaving animal. In addition, a complete understanding requires a knowledge of the intracellular membrane potential changes that regulate the neuronal activity. The technical difficulty of obtaining multiple simultaneous intracellular recordings in an awake rat explains why traditionally these two types of information have been gathered separately by recording extracellularly or intracellularly depending on the desired information. Because the circuitry of transmembrane currents of neurons is completed via the extracellular space, these currents can be measured by electrodes placed outside the cell. The large extracellular negative wave represents the net inward currents that flow during the depolarizing portion of the intracellular action potential. In turn, the extracellular late positive wave represents the net outward currents during the repolarization and afterhyperpolarization of the intracellular action potential.
Our observations suggest that the fine temporal structure of the
extracellular spike contains additional information about the
intracellular action potential. Previous investigators have suggested
that the extracellular unit waveform resembles the first derivative of
the intracellular action potential due to the simple resistive and
capacitive properties of the cell membrane (Brooks and Eccles
1947; Fatt 1957
; Freygang and Frank
1959
; Terzuolo and Araki 1961
). Indeed the shape
of the extracellular initial negative deflection can be approximated by
the first derivative of the intracellular action potential. However,
the late positive component of the extracellular spike is slower than
is expected from the intracellular first derivative. The primary reason
for this discrepancy is the fact that the extracellular spike arises due to the sum of the "leak" current, all active ionic currents and
the capacitive current (Hodgkin and Huxley 1952
). Only
the capacitive current is proportional to the intracellular first derivative. For a small two compartment model, the derivative of the
intracellular voltage and the sum of all currents across the membranes
are equal (Freygang and Frank 1959
). However, previous theoretical work in the olfactory bulb and our own preliminary modeling
data indicate that as the morphological complexity of a simple
compartmental model is increased (i.e., more compartments are added),
the relationship between the intrasomatic voltage derivative and the
summed membrane currents resembles the temporally mismatched
relationship observed in the real data sets (Holt and Koch
1999
; Rall and Shepherd 1968
; our unpublished
observations). These observations suggests that the details of the
later portions of the extracellular spike are influenced by complex
features including neuronal morphology and active conductance
distribution. In addition, the extracellular electrode is sampling
extracellular currents from multiple "compartments" of a neuron due
to volume conduction in the extracellular space. However, as noted in
the preceding text (see Fig. 7), we found that the location of the closest cellular "compartment" along the long axis of the neuron can drastically effect the observed extracellular spike shape.
Unfortunately, the fine temporal correlation between the intracellular
and extracellular waveforms is difficult to quantify for single spikes
due to the fact that there are many sources of background "noise,"
the worst of which is overlapping spikes from other cells
(Harris et al. 2000). However, our data indicate that
relative information about the analogue intracellular action potentials
and membrane polarization can be inferred from averages of the
extracellular spike waveforms (see following text). In addition to
changes in spike amplitude, shape changes in the average intracellular
action potential width correspond to shape changes in average
extracellular spike shape and width. Such a relationship may be useful
when investigating topics such as the modulation of potassium channels
in vivo since action potential width is modulated by a variety of
K+ channels including
IA,
ID,
Ikdr, and
Ic (Storm 1990
).
In addition, these data further validate previous observations that
suggest that the extracellular spike width is a useful measure for the
separation of pyramidal cell and interneuron spikes (Buzsaki and
Eidelberg 1982; Csicsvari et al. 1999a
;
Fox and Ranck 1981
). It is known from intracellular work
that interneurons generally have narrower action potentials than
pyramidal cells recorded under similar conditions (Buhl et al.
1996
; Lacaille et al. 1979
; McBain
1995
; Scharfman 1995
; Sik et al.
1995
). It has been assumed that a similar relationship should
hold for the extracellularly recorded spikes. The data presented here
provide experimental support for this assumption.
Behavioral modulation of the extracellular waveform
Different EEG states are known to be associated with different
patterns of spike activity (Buzsaki et al. 1983). When
we compared the amplitude of extracellular spikes from a single unit
during periods of theta oscillation and nontheta/no-SPW epochs,
we found that there was a significant difference in the average spike
amplitude between these two states. The extracellular spike amplitudes
of both pyramidal cells and interneurons in the pyramidal layer were smaller during theta activity than observed during epochs when theta
oscillations were not present. Since we have also shown that the
amplitude of extracellular spikes varies as a function of membrane
polarization, we conclude that one possible explanation for the
extracellular spike amplitude variation observed during different EEG
states may be differences in the underlying membrane polarization at
the time of the spikes. Although the state-dependent effect on
extracellular spike amplitude was small under the present conditions,
one might expect that when pyramidal cells are strongly activated by
their "place" vectors (O'Keefe and Nadel 1978
;
Quirk and Wilson 1999
), the magnitude of the
intracellular depolarization could be predicted by the amplitude
decrease of the extracellular spikes.
In vivo support for backpropagating dendritic action potentials
The basic temporal relationships between the intracellularly
recorded action potential and the extracellular spike also provide information about the site of action potential initiation in vivo. The
temporal relationship between the peaks of the average extracellular and intracellular waveforms suggest that, on average, the action potential is initiated in the soma and then backpropagates into the
dendrites. These observations do not exclude the possibility of
dendritically initiated spikes during periods of strong synaptic input
such as during sharp waves or epileptic activity (Golding and
Spruston 1998; Kamondi et al. 1998
;
Turner et al. 1991
). In addition, we were able to
estimate the conduction velocity of the backpropagating spike (1.61 m/s) by taking advantage of the known relationship between
intracellular action potential amplitude and somato-dendritic recording
distance (Kamondi et al. 1998
). This number agrees well
with another estimate of the backpropagating action potential
conduction velocity calculated from data collect using linear arrays of
closely spaced recording sites along the somatic and dendritic
compartments of the same neuron (0.5-2.0 m/s) (Buzsaki et al.
1996
).
Sensitivity of tetrodes and spatial clustering techniques
Deciphering the behaviorally relevant computations performed by
neuronal networks requires that a sufficiently large portion of neurons
are monitored simultaneously in the awake animal (Buzsaki et al.
1992; Deadwyler and Hampson 1995
; Skaggs
et al. 1996
; Wilson and McNaughton 1993
).
Current technology uses tetrodes to record groups of neurons near the
tetrode tips whose spikes are then separated off-line by spatial
clustering methods. Our simultaneous intracellular and extracellular
measurements indicate that the amplitude of the extracellular spike
decreases rapidly with distance from the neuron and that extracellular
spikes can be revealed as far as 140 µm from the cell body.
Anatomical experiments estimate that a cylinder with this radius
contains ~1,100 neurons in hippocampal area CA1 (Aika et al.
1994
; Boss et al. 1987
). Although, in theory, all these neurons can be recorded simultaneously by a single tetrode, in practice the number of neurons that are routinely isolated as
separate units is substantially smaller. With the techniques currently
used in our laboratory, the amplitude threshold of easily separable
unit clusters is ~60 µV (Csicsvari et al. 1999a
,b
;
Czurko et al. 1999
; Hirase et al. 1999
).
According to the results of this paper, neurons with amplitudes >60
µV could be recorded from a distance
50 µm. A cylinder with this
radius should contain ~120-140 neurons. Since we observe units as
large as 600 µV (e.g., Fig. 5A), the relationship between
amplitude and distance must be quite steep near the electrode. In fact,
this is likely to be the reason why a tetrode's closely spaced
recordings surfaces show substantially different amplitudes.
The estimated number of clusterable cells in CA1 is several-fold larger
than the number of routinely separated unit clusters by the presently
used methods (range: 1-20 per tetrode) (Csicsvari et al. 1998,
1999a
; Czurko et al. 1999
; Gray et al.
1995
; Harris et al. 2000
; ; O'Keefe and
Recce 1993
; Skaggs and McNaughton 1998
; Skaggs et al. 1996
; Tanila et al. 1997
;
Wilson and McNaughton 1993
). Part of the discrepancy may
be due to localized damage to neurons in the area immediately
surrounding the tetrode. However, at its worst, localized damage can
probably only account for less than half of the "missing" units,
leaving at least 60 separable units still intact. A more
likely potential reason for the large discrepancy between theory and
practice is that the great majority of pyramidal cells are silent in
any given environment. Thompson and Best (1989)
suggested that as many as 63% of the complex cells that they could
record during anesthesia and slow-wave sleep were not active during
active exploration of three different test environments. Our data
suggest that the real number of "silent" or slow-discharging cells
is even higher since our results are based on data collected during
anesthesia or sleep.
The prevalence of silent cells is further supported by examining the
ratio of pyramidal cells to interneurons detected by tetrode
recordings. In general, the observed ratio of simultaneously sampled
interneurons and pyramidal cells is ~1:6 (Csicsvari et al.
1999a; Czurko et al. 1999
; Gothard et al.
1996
; O'Keefe and Recce 1993
; Shen et
al. 1997
; Wilson and McNaughton 1993
). This ratio is five times larger than predicted on anatomical grounds for the
CA1 pyramidal layer (1:33) (Aika et al. 1994
). Since it is commonly believed that interneurons are active in any environment and have a much higher firing rates than pyramidal cells (cf. Freund and Buzsaki 1996
), it is likely that all
interneurons close to the tetrode tip are detected. Therefore the
discrepancy between the physiological and anatomical estimates of
interneuron to pyramidal cell ratios is best explained by a large
number pyramidal cells that are within range of the tetrode but do not
fire sufficient numbers of spikes during the recording period to be
identified as separate cells. Note that this does not mean that these
undetected cells are completely silent, only that they do not fire the
hundreds of spikes necessary to identify them using clustering based
methods. Another important point is that the relative silence of a
large portion of pyramidal neurons favors the statistical separation of
those pyramidal cells that are active. If the majority of pyramidal cells were to be simultaneously active, their separation by the extracellular spike features would be nearly impossible. This logic
predicts that in other brain areas or during periods of strong
activity, when the density of active neurons is high, the presently
available clustering methods will provide unreliable neuron separation
(Harris et al. 2000
).
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ACKNOWLEDGMENTS |
---|
We thank M. Recce for comments on the manuscript and J. Hetke and K. Wise for supplying us with the silicon probes (1P41RR09754).
This work was supported by National Institutes of Health Grants NS-34994, MH-54671, and MH-12403 (to D. A. Henze), the Epilepsy Foundation of American (D. A. Henze), and an Eotvos fellowship (Z. Borhegyi).
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FOOTNOTES |
---|
Address for reprint requests: G. Buzsáki, Center for Molecular and Behavioral Neuroscience, Rutgers University, 197 University Ave., Newark, NJ 07102 (E-mail: buzsaki{at}axon.rutgers.edu).
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 10 December 1999; accepted in final form 6 April 2000.
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REFERENCES |
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