Departmento de Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain
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ABSTRACT |
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López-Aguado, L., J. M. Ibarz, and O. Herreras. Modulation of Dendritic Action Currents Decreases the Reliability of Population Spikes. J. Neurophysiol. 83: 1108-1114, 2000. During synchronous action potential (AP) firing of CA1 pyramidal cells, a population spike (PS) is recorded in the extracellular space, the amplitude of which is considered a reliable quantitative index of the population output. Because the AP can be actively conducted and differentially modulated along the soma and dendrites, the extracellular part of the dendritic inward currents variably contributes to the somatic PS by spreading in the volume conductor to adjacent strata. This contribution has been studied by current-source density analysis and intracellular recordings in vivo during repetitive backpropagation that induces their selective fading. Both the PS and the ensemble action currents declined during high-frequency activation, although at different rates and timings. The decline was much stronger in dendrites than in the somatic region. At specific frequencies and for a short number of impulses the decrease of the somatic PS was neither due to fewer firing cells nor to decreased somatic action currents but to the blockade of dendritic action currents. The dendritic contribution to the peak of the somatic antidromic PS was estimated at ~30-40% and up to 100% at later times in the positive-going limb. The blockade of AP dendritic invasion was in part due to a decreased transfer of current from the soma that underwent a cumulative increase of conductance and slow depolarization during the train that eventually extended into the axon. The possibility of differential modulation of soma and dendritic action currents during APs should be checked when using the PS as a quantitative parameter.
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INTRODUCTION |
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During synchronous action potential (AP) firing of
CA1 pyramidal cells the action currents from individual cells sum in
the extracellular space raising a sharp negative field potential (FP) known as the population spike (PS). Its amplitude increases linearly with the number of firing neurons (Andersen et al. 1971)
and is considered a reliable quantitative index of the population
output. Variations of the PS may, however, be due to factors other than the cell number or the firing synchronism, such as changes in the
amount of current contributed by the firing cells to the extracellular space. In the laminated CA1, the PS is typically recorded at the stratum pyramidale, which is dominated by cell somata, but may also be
contributed by currents arising from activated neuronal elements
nearby, such as dendrites. In fact, the PS negativity spans through the
basal and the proximal apical region, a result that is taken as an
indication for active dendritic AP generation (Fujita and Sakata
1962
; Sperti et al. 1966
). Subsequent
current-source density (CSD) analysis and intradendritic recordings
have definitely demonstrated that CA1 pyramidal cell dendrites can
actively conduct and even initiate the AP (Herreras
1990
; Leung 1979b
; Turner et al.
1991
; Wong et al. 1979
). Because soma and
dendrites can be differentially modulated (e.g., Callaway and
Ross 1995
; Herreras and Somjen 1993
;
Mackenzie and Murphy 1998
; Spruston et al.
1995
), we investigated the extent of the relative dendritic
current contribution to the PS in the s. pyramidale.
For this purpose we calculated the average local currents of somata and
dendrites by CSD and compared its amplitude and waveform with that of
the PS. CSD provides the net local current generated within a small
volume of tissue (Nicholson and Freeman 1975), and in
the strongly stratified CA1 region reflects the sum of transmembrane
currents from analogous specific neuronal subregions (e.g.,
Herreras 1990
; Richardson et al. 1987
).
Therefore, discrepancies in the temporal evolution of FPs and CSDs can
be used to disclose the contribution from neuronal generators located
in different strata. We have unmasked the dendritic contribution using
the selective gradual fading of backpropagated APs during
high-frequency antidromic activation (Lorente de Nó
1947
; Spruston et al. 1995
). A unitary
intracellular study was also made to assess whether individual cells
fired during the protocol.
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METHODS |
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Female Sprague-Dawley rats weighing 200-250 g were anesthetized
with urethane (1.2-1.5 g/kg ip). Surgical and stereotaxic procedures
were as previously described (Herreras 1990). Antidromic activation of the CA1 was achieved by alvear stimuli (0.07-0.1 ms,
0.1-0.5 mA) delivered in short (150 ms) tetani of
300 Hz.
Extracellular recordings and CSD
Two micropipettes (filled with 150 mM NaCl; 3-6 M) connected
to DC-coupled field-effect transistor (FET) input stages were used. One remained stationary in the CA1 s. pyramidale to test the
constancy of the evoked PS amplitude, and another was used to explore
dorsoventral trajectories in 50-µm steps. After filtering (1 Hz to 5 kHz band-pass) and amplification, signals were recorded on VCR, stored
in a computer (20-40 kHz acquisition rate, Digidata 1200, Axon
Instruments, Burlingame, CA), processed by Axotape software (Axon), and
then further analyzed by the Axum program (Trimetrix, Seattle, WA).
Depth profiles of evoked potentials were used for CSD analysis
(Nicholson and Freeman 1975
). At each recording station
six to eight trains of stimuli were delivered 1 min apart. Although in
most cases trains contained 30 impulses, only the first 15 were used
for this study, because the constancy of responses was less reliable in
the second half of the train. Trains were rejected for averaging when
some of their PSs at the stationary somatic electrode differed more
than 10% of those from a grand average made through the entire
profile. Averages at each station had at least four valid trials. A
detailed account of technical and theoretical considerations for the
calculation of CSD in vivo has been presented elsewhere
(Herreras 1990
). We used the customary unidimensional
approach for the calculation of the extracellular currents
(Freeman and Nicholson 1975
). In preliminary experiments
we checked that relative tissue resistivity between strata was
unchanged during the stimulating protocol, so that CSD values are given
in proportional units. Smoothing procedures intended to decrease high
spatial noise were not used, because they introduce substantial
perturbations in the relative amplitude and spatial distribution of
high-frequency components, i.e., action currents (Herreras
1990
).
The PS in the s. pyramidale was measured as the amplitude to the peak of its negative-going limb compared with the precedent baseline. On some occasions, when the artifact partially overlapped the evoked potential, the most positive value between them was used instead. In the stratum radiatum it was measured as the voltage from the negative peak (when discernible) to an imaginary line linking the precedent positive crest and the following inflection point in the rising phase. The precedent positivity is clearly discernible in all but the s. pyramidale, where the negative-going limb departs from baseline. Current sinks were similarly measured. In this case, a sink was present when the inflection point was at a negative value, otherwise a pure source was assumed and the value taken as zero. All values are given as the percentage of control (mean ± SE). Mean values were obtained with six valid FP profiles from different animals.
Intracellular study
Micropipettes (1.5 mm OD) were backfilled with 2-4 M
potassium acetate (60-120 M). Signals were amplified using a bridge circuit amplifier, filtered at 10 kHz, and stored on VCR for later analysis by using pClamp and Axotape computer programs (20-40 kHz
acquisition rate). Recordings were made from electrophysiologically identified pyramidal cells located within the s. pyramidale of the CA1,
identified by the characteristic PS. Cells that did not fire a single
antidromic AP after alvear stimulation were rejected. We considered
healthy cells to be those with resting membrane potential more negative
than
60 mV (
66.3 ± 2.7 mV; n = 7) and overshooting APs. The average apparent input resistance calculated from
the linear range of current voltage (I-V) plots
was 30.2 ± 4.2 M
(range, 22-41 M
). Once stabilized, all
cells fired spontaneous APs at a very low rate (<1 Hz).
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RESULTS |
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Extracellular potentials and subcellular current generators
Antidromic activation raised a sharp negative PS (24.96 ± 0.51 mV) followed by a slower (10-15 ms), small, positive wave at the
level of the s. pyramidale. The PS became a positive-negative diphasic
spike for ~200-250 µm within the proximal s. radiatum (Fig.
1A, FP, see first pulse), and
a pure positive spike at more distal positions, as the negative
component gradually faded off. Within the stratum oriens, the PS
unfolded in a two-spike sequence toward the alveus. The first remained
stationary, whereas the second increased in latency and declined
faster. In the CSD analysis (Fig. 1A, right), the
earliest current was a sink (black areas) at the level of the s.
pyramidale, corresponding primarily to the population inward currents
during synchronous somatic APs. This somatic sink propagated actively
into the apical and basal dendritic trees for ~225 and 125 µm,
respectively, and was always preceded by a passive source (small arrows
in pulse 1, Fig. 1, A and B) corresponding to the
leading outward passive currents depolarizing the membrane in front of
the AP. The peak amplitude at each of the three successive stages below
the s. pyramidale (taken as reference) was 71 ± 4%, 49 ± 4%, and 61 ± 5%, respectively, decreasing thereafter to
extinction (data obtained from n = 19 animals in
another series of experiments). The enlarged tracings in Fig.
1B (left) show the short duration of the sinks at
different strata and their temporal relation to the PS waveform
recorded at the s. pyramidale, whose duration spans all sinks. A
decreasing source was obtained in the region of distal dendrites in the
stratum moleculare, as expected where the AP propagation became
passive. Negative PSs were recorded over a wider dendritic band than
current sinks, because these give rise to negative potentials that
spread in the extracellular space beyond the active membrane region
(Lorente de Nó 1947; Rall 1962
).
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Dendritic action currents contribute to the PS at the s. pyramidale
During repetitive activation, a negative potential envelope
of 2-4 mV developed in the s. pyramidale and s. oriens in parallel with a smaller positive potential in the apical region, except during
the initial impulses that were positive throughout (see inset in Fig. 1A). This slow envelope outlasted
the stimulating period, returning to baseline within 1 s. To
simplify the comparison with the subsequent intracellular study we will
refer mainly to the initial 15 pulses of a train (see
METHODS). As a whole, both the PS and the ensemble action
currents declined during high-frequency alvear stimulation, although
somatic and dendritic regions declined at different rates and timings
(Fig. 1, A and C). The rate of decline was faster
the higher the stimulus frequency and was stronger the farther away
from the cell soma layer. It was also stronger in the basal than in the
apical dendritic tree. Note the selective blockade of the second
negative peak in the uppermost row of the FPs in the s. oriens, whereas
the first peak, attributed to axon initial segment currents
(Sperti et al. 1966), remained unchanged. As an example
in apical dendrites, the PS at 100 µm below the s. pyramidale (ap100)
had decreased by the 15th pulse to 57.6 ± 5.5% and 40.9 ± 4.1% at 100 and 200 Hz, respectively (see Fig. 1C, upward
triangles). As it concerns the ensemble currents, the basal sink (in
the s. oriens) was extremely labile, fading out after a few pulses
(Fig. 1A, right, 5). At 100 Hz, the apical sink
at a location of 100 µm below the s. pyramidale had decreased to
37.8 ± 22% by the 15th pulse, whereas at 200 Hz it had
disappeared entirely below 50 µm of the apical region, remaining only
within a narrow band dominated by the thick apical shafts (Fig.
1A, CSD, and Fig. 1B, pulse 15). Except in this
region, the source-sink sequences were gradually transformed into pure
sources (top and two bottom rows in Fig.
1A, CSD, and enlargements in Fig. 1B, pulse 15),
verifying that shorter distances were progressively invaded by a
regenerative AP, whose spread became passive. It is notable that
negative PSs could be recorded during the train over a somatopetally
shifting dendritic band, whereas the corresponding currents at the
border were already pure sources. This was the expected finding
slightly beyond the farthest activated region and demonstrates that
negative PSs do not necessarily denote active events at the recording
position. The negativity on this boundary zone is caused by the spread
of currents in the extracellular space from the nearest active region,
the so-called volume propagation (see following description and
schematic in Fig. 2D).
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In the s. pyramidale, the PS and the averaged somatic currents evolved
differently (an example is illustrated in Fig. 2A). By the
15th pulse, the PS was always smaller than control (66.4 ± 8%;
see evolution in Fig. 2B, FP, and in Fig.
1C). The inward currents first increased and then decreased
but at lower rate than the PS, remaining at 100.3 ± 7.4%
(measured at the peak) by the 15th pulse (compare 1st and 15th pulses
in Fig. 2B, CSD). In parallel, the duration of the somatic
sink increased because of the gradual fading of a delayed current
source at the positive-going limb of the PS (marked by the small arrows
in Fig. 2, A and C, and the curved arrow in Fig.
2B). This fading source corresponded to the outward passive
currents from the delayed active dendritic sinks (see matching currents
in Fig. 1B). If the somatic currents were measured at the
time of the PS peak instead of their own, they were even larger
(122.5 ± 6.3% by the 15th pulse), increasing further the
divergence with the decreased PS peak. For longer trains (more than 15 impulses), both the PS and the ensemble somatic currents decreased
still further, but once dendritic invasion was minimized, they
decreased at similar rates (not shown).
The decrease of the PS at the s. pyramidale was time related to the missing current contribution from the extracellular dendritic sinks nearby as they faded out. If these events are causally related, the dendritic contribution to the PS recorded at the s. pyramidale should be smaller at the peak than at the rising phase, because dendritic inward currents are slightly delayed (see Fig. 1, A and B). In fact, when the temporal course of the population currents and FPs were compared, it was evident that the PS peaked later than the current sink, and most important, the PS negativity lasted much longer (Fig. 2C, 1st pulse). From the instant when the somatic sink ended (i.e., when the CSD value becames positive; see arrowhead in Fig. 2C), the negativity of the PS (shaded area) could be entirely attributed to extracellular current spread from dendritic generators (see temporal correlation in Fig. 1B). On the contrary, in the 15th pulse, the time course of currents and potentials in the s. pyramidale was almost identical (Fig. 2C, 15th pulse), indicating that the delayed contributors to the PS negativity (dendritic sinks) had disappeared (Fig. 1, A and B). A moderate contribution from the still-invaded proximal-most apical shafts is very likely. That is, the PS at the s. pyramidale faithfully reflected the evolution of the local (somatic) currents only when it was not contributed by distant generators.
The PS decrease at the cell body layer is not caused by fewer firing cells
The PS decrease in the s. pyramidale in spite of the maintained or
increased population somatic inward currents could also be due to fewer
firing cells, individually contributing more current. To check this
possibility, we made intracellular recordings from seven pyramidal
cells located within the s. pyramidale. Single alvear pulses triggered
an AP followed by a fast inhibitory postsynaptic potential (IPSP)
lasting ~150 ms. During repetitive antidromic activation (Fig. 3,
A and B,
bottom) the AP initially decreased in amplitude and
half-width, and then, increased much more in half-width, became
delayed, and decreased the rate of rise, revealing an AB break
(small arrow in Fig. 3B). Eventually, it failed to invade. Occasional AP failures occurred in some of the initial pulses,
coinciding with the highest rate of conductance increase during the
IPSP (Fig. 3B). These changes were more marked and required fewer pulses the higher the frequency. During the course of
the train, a gradual depolarization with increased conductance developed (arrow in Fig. 3A, top) that
outlasted the stimulating period (double-headed arrow). Its amplitude
varied considerably between cells (30 mV) and was not totally
inactivated, because often some spontaneous APs appeared at the top,
beyond the stimuli (not shown). The duration was ~1 s and roughly
matched the extracellular sustained negative potential recorded in the
s. pyramidale (see inset in Fig. 1A)
Overall, pyramidal cells followed longer trains the lower the
frequency. Regarding the specific windows used for the CSD study (first
15 impulses), all neurons followed activation at 100 Hz for at least
300 ms (i.e., 30 pulses; see an example of 45 pulses in Fig.
3A). At 200 Hz, they could follow trains up to 150 ms
long (30 pulses at 5-ms intervals, Fig. 3B), except two
(of 7) that began to fail in the second half of the train (beyond pulse
15). All seven cells showed AP failures for longer train duration,
i.e., outside the window where a dendritic contribution to the somatic
PS has been shown. Small all-or-nothing spikes (~25 and ~10 mV, see
arrows 1 and 2 in Fig. 3C at 300 Hz) attributed to the
axon initial segment and first node were visible in most neurons after
the AP failed to invade the cell soma. Eventually, the AP block
progressed into axonic regions as these spikes gradually disappeared as
well. Regarding the relevant first 15 pulses at 200 Hz, when
discrepancies between the PS and CSD amplitudes were found, all seven
cells followed regularly. Therefore, the decrease of the PS amplitude
was not caused by fewer firing cells.
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DISCUSSION |
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The most relevant result of this study is that the PS recorded in
the s. pyramidale was significantly attributable to dendritic action
currents during backpropagation of APs. Because of the obligatory delay
of dendritic action currents during antidromic activation, this
contribution was larger at later timesi.e., during the positive-going
limb of the PS waveform. In this phase it could reach up to 100%,
because the net somatic sink terminated well before the negativity of
the PS. From a practical point of view, it is the dendritic
contribution at the time of the PS peak that may be of more interest. A
higher limit could be set at ~30-40%, which constituted the maximum
divergence between the evolution of the ensemble somatic currents and
the PS peak. Once dendritic regenerative AP invasion was blocked, the
somatic PS reflected only the evolution of somatic action currents,
with some contribution of the initial portion of the apical shafts.
After the chosen window of 15 pulses, a further PS decrease may be
accounted for by a decreased number of firing cells and/or a decreased
unitary current contribution, making a unitary study mandatory.
Therefore, the possible modulation of active dendritic properties
should be taken into account when interpreting changes of the PS
measured at a single point in the s. pyramidale.
An interesting observation is that at least part of the longer duration
of the somatic sink was caused by the fading of a delayed current
source. These outward currents corresponded to the return currents
matching the active dendritic sinks (Fig. 2D, arrows marked
a), because both disappeared in parallel. Those more delayed were
responsible for the net source marked by an arrow in Fig. 2C
(the subsequent longer source corresponded to IPSP currents), whereas
those corresponding to more proximal locations overlapped in time with
the late phase of the somatic sink and caused its partial cancellation
and shortening. A fraction of the return outward current constituted
the depolarizing front of propagating APs (arrows at b, Fig.
2D), but obviously, a major fraction left the membrane
locally and through the adjacent active sites of greatly increased
conductance (arrows at a, Fig 2D). This observation
illustrates the strong interaction between adjacent membranes during
active AP propagation, acting as reciprocal shunts. An example of this
reciprocal cancellation can be appreciated in the currents of somata
and proximal apical shafts shown in Fig. 1B (pulse 15). Note
how in spite of the increased duration of the somatic sink due to the
disappearance of delayed sources a positive hump leaving reduced
amplitude is observed that matches the time course of the remaining
lower ap50 sink (small arrows). In fact, it can be easily demonstrated
using computational methods that the somatic transmembrane current is
much larger and longer in passive-dendrite neurons (Varona et
al. 1998). Although this was barely reflected in the
intracellular somatic AP waveform, it had a strong impact on the
extracellular fields, as shown in this study on an experimental basis.
We have shown that individual pyramidal cells can follow short
periods of activation at high frequencies. Even higher rates have been
reported using extracellular recordings (Leung 1979a) in
the same in vivo preparation. The lower efficiency in our experiments may have been due to the mechanical stress and somatic shunt caused by
the intracellular electrode.
As previously reported, the blockade of dendritic AP backpropagation
during repetitive activity is in part due to prolonged sodium channel
inactivation (e.g., Colbert et al. 1997; Jung et al. 1997
). Some of the present data point to a major role of
somatic mechanisms at higher frequencies. The interstimulus interval
fell well within the AP partial refractory period, so that cumulative sodium channel inactivation accounts for the gradual AP change before
its eventual failure. These changes were stronger and faster than those
obtained at the lower frequencies used to study the attenuation of
backpropagating spikes. The slowing in the rising phase of the AP was
most likely caused by a decreased inward current (e.g., see Fig. 1 in
Jung et al. 1997
), which should cause the decrease of
the somatic sink. In fact, this was the overall trend (note the slower
rate of increase in Fig. 2B), although the uncovering of
late phases (see earlier description), and the coincidence of an
initial distinct increment for the first three to eight impulses,
slowed the rate and timing of the decrease.
The AP changes are time related to the extracellularly observed
dendritic AP block and to the somatic cumulative depolarization with
increased conductance (Fig. 3A). The nature of this somatic depolarization was not investigated; reversed IPSP, extracellular potassium accumulation or overlapping depolarizing afterpotentials are
among the possible causes. However, it matched the extracellular slow
potential that was negative in the somatic-basal region and positive in
the apical tree, a dipole configuration typical of active inward
somatic currents. A similar result has long been known to occur during
tetanic antidromic activation of some brain regions and is taken as
evidence for cumulative depolarization of somata but not of dendrites
(e.g., Lorente de Nó 1947), the first of which was
shown in these experiments.
The present results are compatible with a gradually shorter dendritic AP invasion due to reduction of depolarizing currents entering dendrites from the soma, rather than a putative smaller excitability of the apical dendrites, although this also contributed. Such a gating function of the soma may result from the cumulative inactivation of sodium channels and the increased shunt during the slow depolarization. In fact, our results show that the AP invasion block progressed into the axon initial segment, probably as the somatic depolarization extended into this region. In short, the increasing evidence of local modulation of somatodendritic properties not only enhances our knowledge of the repertoire of integrative properties, but it also demands a continuous reevaluation and refinement of the methods used to explore them.
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ACKNOWLEDGMENTS |
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We thank M. J. Yagüe for technical assistance.
This work was supported by grants 8.5/15/98 from the Comunidad Autónoma de Madrid and PB97/1448 from the Spanish Dirección General de Investigación Científica y Técnica.
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FOOTNOTES |
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Address for reprint requests: O. Herreras, Dept. Investigación, Hospital Ramón y Cajal, Ctra. Colmenar km 9, 28034 Madrid, Spain.
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 21 July 1999; accepted in final form 11 October 1999.
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REFERENCES |
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