Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195-7290
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ABSTRACT |
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Oakley, J. C., P. C. Schwindt, and W. E. Crill. Initiation and Propagation of Regenerative Ca2+-Dependent Potentials in Dendrites of Layer 5 Pyramidal Neurons. J. Neurophysiol. 86: 503-513, 2001. The initiation and propagation of dendritic Ca2+-dependent regenerative potentials (CDRPs) were investigated by imaging the Ca2+-sensitive dye Fluo-4 during whole cell recording from the soma of layer 5 pyramidal neurons visualized in a slice preparation of rat neocortex by the use of infrared-differential interference contrast microscopy. CDRPs were evoked by focal iontophoresis of glutamate at visually identified sites 178-648 µm from the soma on the apical dendrite and at sites on the basal dendrites. Increases in [Ca2+]i were maximal near the site of iontophoresis and were graded with iontophoretic current that was subthreshold for evoking CDRPs. CDRP initiation was associated with a [Ca2+]i rise that differed from a just-subthreshold response in both magnitude and spatial extent but whose amplitude declined both proximal and distal to the iontophoretic site. These [Ca2+]i rises, whether associated with subthreshold or regenerative voltage responses, were minimally affected by blockade of N-methyl-D-aspartate receptors but were abolished by Cd2+, suggesting that Ca2+ influx through voltage-gated channels caused the rise of [Ca2+]i. On the assumption that the rise of [Ca2+]i during a CDRP marks the spatial extent of regenerative Ca2+ influx, we conclude that CDRPs can be evoked at any point on the main apical or basal trunk where membrane potential reaches CDRP threshold rather than at discrete "hot spots," the CDRP is initiated at a spatially restricted site, and it propagates decrementally both distal and proximal to its initiation site. These results raise the possibility that synaptic integration may occur first in the dendrites to evoke a CDRP. Because these responses propagate decrementally to the soma, they are able to sum with input from other regions of the cell so that the cell as a whole remains integrative.
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INTRODUCTION |
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In the classic view of
synaptic integration, synaptic currents generated in the dendrites are
summed at a region downstream from the soma to evoke the spike output
of the cell. In this model, the output of the cell is determined by the
sum of each synaptic input weighted by its distance from the downstream
integration point. More recently it has been shown that depolarization
of the apical dendrite of layer 5 neocortical neurons by focal
iontophoresis of glutamate (Schwindt and Crill 1997,
1999
), by current injection through a dendritic recording
electrode (Kim and Connors 1993
; Larkum et al.
1999
; Schiller et al. 1997
), or by synaptic
stimulation (Kim and Connors 1993
; Larkum et al.
1999
; Schiller et al. 1997
; Schwindt and
Crill 1998
) can evoke Ca2+-dependent
action potentials. These Ca2+-dependent
regenerative potentials (CDRPs) have been proposed as a mechanism both
to amplify (Schiller et al. 1997
; Schwindt and
Crill 1997
) and to limit (Oakley et al. 2001
)
the distal synaptic input that reaches the soma. If CDRPs are evoked
locally by dendritic depolarization, there are at least two (and
potentially many) sites of synaptic integration, one near the soma and
the other(s) in the dendrites. According to this idea, synaptic
integration would be determined in part by the number and the spatial
extent of the site(s) of initiation of the regenerative dendritic
potentials. If the dendritic regenerative responses remain localized,
the downstream summing site may still integrate the whole cell current to evoke the cell's Na+ spike output, and the
cell as a whole would remain integrative.
There is both direct (Larkum et al. 1999;
Schiller et al. 1997
) and indirect (Schwindt and
Crill 1997
) electrophysiological evidence, as well as evidence
from Ca2+ imaging (Markram et al.
1995
; Schiller et al. 1997
), that the CDRPs are
not propagated actively to the soma, but several questions remain. Are
all portions of the dendrite capable of generating these regenerative
potentials or only a few regions or only one region? Experiments
employing Ca2+ imaging techniques (Markram
and Sakmann 1994
; Markram et al. 1995
; Schiller
et al. 1995
; Yuste et al. 1994
) suggest that
Ca2+ channels exist over most or all of the
apical dendrite, but other studies have suggested that CDRPs are evoked
only at a few specific points ("hot spots") on the apical dendritic
(Reuveni et al. 1993
) or only in the distal apical tuft
(Schiller et al. 1997
). All available evidence is
consistent with the decremental propagation of the CDRPs toward the
soma, but the spatial extent of the spike-generating region(s) is not
clear. It is not known, for example, if CDRPs are actively
backpropagated. Because they are caused by Ca2+
influx, the backpropagation of these potentials might alter the efficacy of active distal synapses, which are known to be modulated by
a rise of [Ca2+]i
(Neveu and Zucker 1996
; Yang et al.
1999
). In the present study, we sought to answer these
questions by the use of glutamate iontophoresis to depolarize sites on
visualized dendrites of layer 5 neocortical neurons and the imaging of
the Ca2+-sensitive dye, Fluo-4, which was loaded
into the cell through the soma patch electrode. Since the CDRPs are
known to depend on Ca2+ influx through
voltage-gated Ca2+ channels, we measured changes
in Fluo-4 fluorescence to determine the spatial-temporal increase in
dendritic Ca2+ concentration
([Ca2+])i during CDRPs.
We deduced the mode of initiation and propagation of CDRPs from the
changes in [Ca2+].
The use of iontrophoresed glutamate to depolarize a visualized
dendritic site is convenient for the several reasons discussed in
Oakley et al. (2001). Foremost, it allows the amplitude
and duration of long-lasting dendritic depolarization to be controlled conveniently by the experimenter at a precisely known distance from the
soma. Importantly for the present study, it allows the response to the
same depolarizing stimulus to be compared in the absence and in the
presence of channel-blocking agents that would alter or block synaptic transmission.
In the study by Schwindt and Crill (1999), short
iontophoretic pulses of glutamate evoked transient
Ca2+ spikes that lasted ~100 ms, while longer
iontophoretic pulses evoked a long-duration Ca2
spike (a "plateau") that persisted for the duration of the
iontophoretic current. In this study, we focused on the rise in
[Ca2+]i during the
regenerative plateau response in part because these long-lasting
plateaus have been less well studied than the transient Ca2+ spikes. A great experimental advantage of
the plateau is that it lasts as long as the iontophoresis, which is
under the experimenter's control. A long-lasting response allows the
use of a sufficiently long CCD camera integration time to obtain a
fluorescence signal with a satisfactory signal-to-noise ratio while
also allowing a sufficient number of images to be obtained to define
adequately the time course of the
[Ca2+]i transient.
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METHODS |
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Tissue preparation and solutions
Sprague-Dawley rats of either sex aged 20-32 days postnatal
were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg) and
killed by carotid section. Following craniotomy, a section of dorsal
frontoparietal (sensorimotor cortex) was removed, fixed with
cyanoacrylite glue to the stage of a microslicer, and submerged in
ice-cold physiological saline solution (PSS) containing (in mM) 130 NaCl, 3.0 KCl, 2.0 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, and 20 glucose gassed with 95% O2-5%
CO2 (carbogen). Coronal slices (300 µm) were
cut and stored in a holding chamber filled with carbogenated PSS at
34°C. Individual slices were transferred to a recording chamber where
they were maintained submerged in 29-32°C carbogenated PSS flowing
at >2 ml/min. In some experiments TTX (1 µM, Sigma Chemical, St.
Louis, MO) or D()-2-amino-5-phosphonopentanoic acid
(AP-5) (100 µM, Precision Neurochemicals, Vancouver BC, Canada) were
added to the PSS, or 200 µM CdCl2 was
substituted for an equimolar amount of CaCl2 and
NaH2PO4 was omitted to
avoid precipitation.
The bottom of the 20-mm diam, 3-mm-deep recording chamber was formed by a No. 1 glass coverslip. The slice was held in place on the coverslip by thin glass bars straddling the two arms of a U-shaped platinum wire 300 µm thick. The gravity-fed inflow and suction outflow were positioned to promote laminar flow across the slice. The recording electrode and the iontophoretic electrodes opposed each other so that they could be placed independently anywhere in the chamber.
Recording methods
An upright microscope (Olympus BX50WI) fit with ×40 LWD water-immersion objectives and DIC optics was used to view cells in the top 100 µm of the slice. The slice was transilluminated using infrared (Omega 770 ± 40 nm band-pass filter) and viewed using a CCD camera (Hamamatsu) and a high-resolution video monitor (Sony). The microscope was fixed to an x-y translation stage that allowed the microscope to be positioned independently of the recording and iontophoretic electrodes so that different portions of the cell could be viewed without disturbing these pipettes.
Micropipettes pulled from VWR 75 ml capillary glass and filled with
standard intracellular solution containing (in mM) 135 KMeSO4, 5 KCl, 2 MgCl2, 10 (3-[N-morpholino]propanesulfonic acid) (MOPS), 0.1 ethylene glycol-bis(-aminoethyl
ether)N,N,N',N'-tetraacetic acid (EGTA), 2 Na-ATP, 0.2 Na-GTP, 0.15 Fluo-4 (Molecular Probes) and (in %wt/vol) 0.01 Lucifer
yellow, K+ salt (Molecular Probes) were used to
make whole cell recordings.
Extracellular DC resistance of the pipettes was 2-4 M. The seal
resistance formed with the soma membrane was >1 G
before break in
to the whole cell configuration. Series resistance in the whole cell
configuration was monitored by maintaining bridge balance during a
hyperpolarizing current pulse. Recordings were discarded if series
resistance became >50 M
.
An Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) was used in bridge mode to record somatic membrane potential and inject current through the recording pipette. Stable recordings lasting 1-2 h were frequently obtained. Resting potential was taken as the difference between intracellular and extracellular potentials recorded on a strip chart recorder. Recorded potentials were corrected for a tip potential of 10 mV. Current and voltage recordings were filtered at 10 kHz and stored on videotape using pulse code modulation (Neurodata) and were later digitized (Axon Instruments TL-1 125 interface) and analyzed with the program WCP written and distributed by John Dempster and with the program IGOR (WaveMetrics, Lake Oswego, OR).
Glutamate iontophoresis
Fluorescence microscopy was used to simultaneously view the
dendritic arbor and the iontophoretic electrode so that glutamate could
be applied to visually identified segments of dendrite at a known
distance from the soma. After obtaining a stable whole cell recording,
short, hyperpolarizing current pulses (<500 pA) were applied to the
recording pipette to iontophorese K+-Lucifer
yellow and Fluo-4 dyes into the cell. The DIC analyzer was removed, and
the light path was switched from transmitted light to epifluorescence
illumination with excitation at 400 nm and emission at 535 nm.
Illumination at 400 nm was chosen to excite K+-Lucifer yellow dye without bleaching Fluo-4
dye (peak excitation 485 nm).
Iontophoretic electrodes were pulled from 1 mm OD glass capillary
tubes. Their tips were broken to 2 µm diam, and they were filled with
(in mM) 150 glutamatic acid, 2.24 CaCl2, 4 KCl,
30 N-(2
hydroxyethyl)piperizine-N'-(2-ethanesulfonic acid) (HEPES), and (as % wt/vol) 0.05% K+-Lucifer yellow salt,
pH 7.4. The second current amplifier and headstage of the Axoclamp-2A
amplifier was used to provide positive DC holding current (+5 nA) and
negative iontophoretic pulses (1-2 s duration, 5- to
100-nA
amplitude). Once the dendrites contained sufficient dye, the
iontophoretic electrode was visually guided to a site on the dendritic
tree. Movement of the electrode tip over a range of 10-20 µm near
the visualized dendrite caused the iontophoretically evoked response to
vary from zero to maximal. The glutamate concentration 10 µm from the
electrode tip caused by an iontophoretic current sufficient to evoke a
plateau potential was estimated to be
10 mM (Oakley et al.
2001
).
Imaging methods
Once the iontophoretic electrode was placed in the dendritic arbor and a stable iontophoretic response was obtained, the excitation wavelength was switched to 508 nm which was found to excite the Fluo-4 dye but not the K+-Lucifer yellow dye in the cell or in the iontophoretic electrode. Fluorescence signals were recorded at ×40 magnification. Imaging commenced >10 min after break-in to allow Fluo-4 to diffuse from the patch solution into the dendrites. Excitation wavelength was controlled by a TILL system (Photonics, Planeg, Germany). When not imaging, the excitation light was stepped to a long wavelength, and the scattered light from the TILL system was blocked by a high-pass filter. In most experiments, the iontophoretic electrode was centered within the imaged region. In some experiments, the imaged region proximal or distal to the iontophoretic electrode was increased by placing the iontophoretic electrode near one end of the imaged region. In those cells the spatial extent of the fluorescence signal was only measured in one direction from the iontophoretic electrode (the unmeasured direction is indicated by n.m. in Fig. 4).
Prior to the iontophoretic pulse, 10 images of resting fluorescence (FR) were acquired using a Pentamax cooled CCD camera (Princeton Instruments, EEV 1,024 × 512 chip) in frame transfer mode and Metafluor software (Universal Imaging). The first image contained no data and was thrown out. An additional 40 images were acquired (50 ms each) during and following the iontophoretic pulse. These images, taken at ×40, were acquired using a subchip defined around the dendrite with pixels binned by 3 to decrease the acquisition time required to give a satisfactory signal-to-noise ratio. Following each experiment images also were taken at ×10 using K+-Lucifer yellow excitation (425 nm) to define the straight-line distance from the center of the soma to the iontophoretic site during off-line analysis.
The ×40 image was used to define regions of interest (ROIs) that
consisted of serial 8.5-µm-long sections of dendrite and that were
analyzed off-line. The average fluorescence of each ROI was calculated
with the use of the software package Metafluor. A shading image was
acquired in a weak dye solution (which provided a bright, uniform
fluorescence) for each subchip and binning combination employed. The
raw fluorescence signals were corrected for shading by dividing each
ROI by the value of the fluorescence at the same ROI in the shading
image. The fluorescence signals were corrected for bleaching by fitting
a line to the fluorescence of the nine frames taken at rest prior to
the stimulus. The fitted line was then extrapolated and subtracted from
the shading corrected fluorescence signals. After the application of
these shading and bleaching corrections, the stimulus-linked change in
fluorescence (F) in each ROI were computed as
F = FS
FR, where
FS is the average fluorescence of the
ROI following stimulation and FR is
the prestimulation resting fluorescence of the ROI. Data are presented
as the relative change in fluorescence,
F/F = (FS
FR)/(FR
FB) where
FB is the background fluorescence
which was determined from an ROI away from the fluorescent dendrite.
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RESULTS |
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Cell properties
Recordings were made from 27 neocortical layer 5 pyramidal neurons
in slices from 20 rats 20-32 days old. Resting potentials ranged from
54 to
79 mV (mean:
72 mV). Input resistance, measured with long
hyperpolarizing current pulses, varied from 24 to 100 M
(mean: 45 M
). Cells were accepted for analysis if they had stable resting
potentials and fired repetitive action potentials to depolarizing soma
current. All recorded cells exhibited regular spiking (Connors
and Gutnick 1990
) in response to depolarization of the soma by
injected current. No intrinsic bursters or fast spiking cells
(Connors and Gutnick 1990
) were recorded.
Plateau properties
In a previous study Schwindt and Crill (1999) found
that a long-duration, all-or-none, Ca2+-dependent
action potential (a "plateau"), which was usually preceded by an
initial, transient, Ca2+ spike, could be evoked
by focal iontophoresis of glutamate on the apical dendrite of layer 5 pyramidal cells. Similar responses were evoked by 1- to 2-s duration
iontophoresis of glutamate in this study, which employed whole cell
patch recording methods and direct visualization of the soma and
dendritic tree (see METHODS). The properties of these
responses, as recorded using whole cell, IR-CCD methods, are described
in detail in Oakley et al. (2001)
. Figure
1 illustrates some key features of the
Ca2+ spike and plateau. In this cell
iontophoresis (
60 nA) at a site 460 µm from the soma on the apical
dendrite evoked an initial, low-threshold, slow, action potential on
which was superimposed a higher-threshold, fast spike (Fig. 1, black
trace). The different time courses and thresholds of the
fast and slow spikes are not obvious at the slow sweep speed used in
this figure (cf. Schwindt and Crill 1997
). Similar fast
spikes were eliminated by TTX application, and similar slow spikes were
eliminated by Cd2+ application (see following
text). The membrane potential response during the remainder of this
iontophoresis remained subthreshold for action potential initiation. A
slightly larger iontophoretic current (
70 nA) again evoked the
initial Ca2+ and Na+
spikes, which were then followed by a long-duration action potential (the plateau) that repolarized only when the iontophoretic current was
terminated (Fig. 1, green trace). In all cells tested, a larger iontophoretic current (
80 nA in Fig. 1) decreased the latency to
plateau initiation but did not increase plateau amplitude (Fig. 1, red
trace). Similar plateaus were evoked by focal iontophoresis of
glutamate at 22 identified sites on the apical dendrite in different
experiments.
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Location and properties of evoked [Ca2+]i increase in the apical dendrite
Since the plateau depends on Ca2+ influx
through voltage-gated channels (Oakley et al. 2001;
Schwindt and Crill 1999
), its site of initiation and its
propagation along the dendrite was expected to be indicated by the
temporally correlated rise of intracellular Ca2+
concentration ([Ca2+]i).
The rise of [Ca2+]i was
estimated by measuring the change in relative fluorescence (
F/F, see METHODS) of the
Ca2+-sensitive dye Fluo-4 during glutamate
iontophoresis. An increase of
F/F signals an
increase in [Ca2+]i.
Fluorescence responses (
F/F) were measured and
compared during iontophoreses that were subthreshold and suprathreshold
for plateau initiation, both in physiological saline and in the
presence of various blocking agents.
Typical results obtained in physiological saline are illustrated in
Fig. 2, which is from the same cell as
shown in Fig. 1. As indicated in the schematic drawing of Fig.
2A, the field of view was centered near the visually
identified position of the iontophoretic electrode. In the cell of Fig.
2, this visualized region of the apical dendrite extended from 290 to
540 µm from the soma (indicated by red box in Fig. 2A).
The visually identified location of the iontophoretic electrode is
indicated by the dashed arrow in Fig. 2A. Images were taken
every 50 ms, starting 500 ms before the iontophoretic pulse (to
establish the resting fluorescence level) and ending 1 s after the
iontophoretic pulse was terminated. The entire visualized length of the
apical dendrite was divided into contiguous 8.5-µm-long segments in
which average F/F values were measured (see
METHODS).
F/F values are
represented according to the pseudocolor scale at the bottom
left of Fig. 2B. In the pseudocolor plot of Fig.
2B, and in all other such plots in this paper,
F/F is plotted as a function of both distance
along the apical dendrite (plot ordinate) and time after the start of
the sweep (plot abscissa) to show the spatial-temporal increase in
F/F during an iontophoresis. Note that the
ordinate in this (and all other such plots) starts at the most proximal
part of the imaged region rather than at the soma. The membrane
potential response (recorded at the soma) during the iontophoresis is
shown at the same time scale below the pseudocolor plot (Fig.
2B, red trace). This is the same response to the
60 nA
iontophoresis shown in Fig. 1. From the pseudocolor plot, it can be
seen that the largest rise in
[Ca2+]i occurred at the
iontophoretic site (indicated by dashed line on pseudocolor plot).
During the initial Ca2+ and
Na+ spikes,
[Ca2+]i increased above
resting levels at locations both proximal and distal to the
iontophoretic site. Following these initial spikes, [Ca2+]i decayed to the
resting level at these proximal and distal locations, whereas
[Ca2+]i remained above
the resting level near the iontophoretic site for the remainder of the
iontophoresis. After the iontophoresis was terminated,
[Ca2+]i decayed to
baseline. In this and all cells examined, an iontophoresis that was
subthreshold for either the plateau or the initial transient Ca2+ spike caused an increase of
[Ca2+]i, and the largest
rise in [Ca2+]i occurred
near the iontophoretic site.
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In Fig. 2C, the plateau was evoked (red trace at
bottom) during a larger (70 nA) iontophoresis at the same
site. As indicated by the pseudocolor plot (Fig. 2C,
top), during and immediately after the initial
Ca2+ and Na+ spike the
spatial-temporal increase of
[Ca2+]i was similar to
that of Fig. 2B. Subsequently, when the plateau was evoked,
the increase of [Ca2+]i
at the iontophoretic site was larger than during the subthreshold response of Fig. 2B or the subthreshold portion of the
response that preceded the plateau in Fig. 2C. In addition,
[Ca2+]i rose above its
rest value both proximal and distal to the iontophoretic site following
plateau initiation, and this rise persisted for the duration of the
plateau. Thus the plateau was associated with a
[Ca2+]i rise that
differed from a subthreshold response in both magnitude and spatial extent.
Our primary finding is that the rise of [Ca2+]i associated with plateau generation declined with distance from the iontophoretic spike, both proximally and distally. This same pattern, highest around the iontophoretic site and lower at the proximal and distal boundaries of the imaged region, was evoked during plateau generation in all recorded cells. Assuming for the moment that the rise of [Ca2+]i is caused entirely by Ca2+ influx through voltage-gated channels, these results imply that dendritic membrane potential (i.e., plateau amplitude) also declined with distance. That is, the plateau propagates decrementally both proximal and distal to its site of initiation. In contrast, a regenerative response that propagated actively with constant amplitude, like a Na+ spike propagating down the axon, would be expected to result in a similar rise of [Ca2+]i along the whole region of active propagation. The assumptions on which this conclusion is based were tested in experiments described in the following text.
Because [Ca2+]i also rose
during a subthreshold response and because
[Ca2+]i did not decline
abruptly with distance from the iontophoretic site during either the
subthreshold response or the plateau, the precise spatial extent of
active plateau generation (i.e., the region where net inward ionic
current caused a regenerative response) was difficult to determine.
Since subthreshold responses evoked a rise of
[Ca2+]i, the amplitude of
even a decrementally propagating plateau would be great enough to
activate Ca2+ channels to cause a nonregenerative
Ca2+ influx and a rise of
[Ca2+]i. To provide some
quantitative estimate of the spatial extent of the active region (in
this and all recorded cells), a contour line was drawn around the
region in which F/F was
50% of the peak
F/F reached during the plateau (Fig.
2C, black line). This 50%-of-peak contour line was used to
estimate the region of active plateau initiation.
Our rationale for adopting the 50%-of-peak contour line as an index of
the spatial extent of the active plateau generating region is
illustrated by the plots of Fig. 3. The
plot of Fig. 3A is a more conventional representation of the
florescence data of Fig. 2C. Florescence at three times
during the response (at rest, 250 ms; during the subthreshold response
after the initial Na+ and
Ca2+ spikes, 1,000 ms, and during the plateau,
1,500 ms) is plotted versus distance from the soma over the field of
view. Although we interpret the fluorescence data as resulting from
variations of dendritic membrane potential, it should be realized that
the relation between fluorescence signal (F/F)
and dendritic membrane potential is quite indirect and complex. Because
of the sublinear nature of the binding relation between
Ca2+ and this high-affinity dye, a small
F obtained when ambient [Ca2+]i is high may
represent a larger increment of
[Ca2+]i than does a
larger
F obtained when ambient
[Ca2+]i is low, and of
course there is a highly nonlinear relation between membrane potential
and Ca2+ current. Thus no simple or quantitative
conclusions can be drawn from the plots of Fig. 3A about the
variation of [Ca2+]i or
dendritic membrane potential beyond the fact that both quantities must
be larger where the fluorescence signal is larger (again assuming the
signal arises solely from Ca2+ influx through
voltage-gated channels). During the plateau in Fig. 3A, the
fluorescence signal rose significantly above the subthreshold response
over most of the 250 µm field of view, but the rise was particularly
abrupt over the region bracketed by the vertical dotted lines,
suggesting that Ca2+ influx was particularly
large and relatively uniform over this region. We assume therefore that
this region constitutes the extent of active plateau generation, and
the vertical dotted lines represent the 50%-of-peak contour lines of
Fig. 2C at this point in time.
|
The plot of Fig. 3B is from a different cell in which
several subthreshold responses were obtained from iontophoresis 280 µm from the soma before evoking a plateau. The rightmost data point
(labeled "plateau") is the average F/F
value measured over the first 200 ms (the 1st 4 images) of the plateau
at the iontophoretic site. The data points to the left were obtained
from the same region and over the same time interval but during smaller
iontophoretic currents that evoked only the subthreshold responses. The
subthreshold signals were graded with iontophoretic current as
indicated by the line fitted to the three subthreshold data points. The
average
F/F value measured during the plateau
lies significantly above this line, suggesting there was a "jump"
in [Ca2+]i after the
plateau was triggered. Such a jump is expected because of the
regenerative Ca2+ influx associated with the
initiation of the plateau. The horizontal dashed line in Fig. 3 is
drawn at 50% of the peak
F/F value. We assume
F/F values that lie above this arise from
active (regenerative) plateau initiation, whereas values below the are
ascribed to nonregenerative Ca2+ influx.
Based on the observations and rationale described in the preceding
text, the proximal and distal extent of the active plateau-generating region was defined as the most proximal and distal extent of the 50%-of-peak contour line during a plateau. Using this criterion, the
active plateau-generating region was remarkably small but of similar
magnitude among the cells tested. Active plateau initiation extended 50 µm proximally and 38 µm distally from the iontophoretic site in the
experiment of Fig. 2C. Plateaus were evoked on the apical
dendrite of 12 cells bathed in physiological saline. Data from these 12 plateaus are plotted as squares in Fig.
4. The squares mark both the distance of
the iontophoretic site from the soma in each cell and the distance
where F/F during the plateau was maximal,
since both distances were identical. An estimate of the spatial extent
of active plateau initiation in each cell (measured using the
50%-of-peak contour described in the preceding text) is indicated by
the length of the vertical bars through each data point. In these
experiments, the proximal extent of active initiation ranged from 20 to
113 µm (mean: 63.2 µm), and the distal extent ranged from 10 to 207 µm (mean: 51.4 µm). The proximal and distal extents were not
significantly different (P = 0.47, paired, 2-tailed, Student's t-test).
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The plot of Fig. 4 also shows that plateaus were evoked over the whole extent of the apical dendrite examined (from 178 to 648 µm from the soma), and all plateaus were centered spatially about the iontophoretic site. The ability to evoke plateaus at sites over this length of the apical dendrite indicates that Ca2+ channel density is adequate for plateau generation over (at least) this length of the apical dendrite.
Increased [Ca2+]i results primarily from voltage-gated Ca2+ channel activation
It was assumed in the preceding text that the rise in [Ca2+]i is caused by influx of Ca2+ through voltage-gated Ca2+ channels. However, Ca2+ might also enter through N-methyl-D-aspartate (NMDA)-sensitive glutamate channels. The increase of [Ca2+]i observed during dendritic depolarizations that were subthreshold for plateau initiation (e.g., Figs. 2, A and B, and 3) particularly raised the possibility of Ca2+ influx through NMDA-sensitive channels.
To investigate the source of the Ca2+ responsible
for the rise in [Ca2+]i,
the spatial extent of plateaus evoked at the same site was investigated
before and after the addition of 100 µM of the specific NMDA receptor
antagonist AP-5 to physiological saline in two cells. In one cell, the
extent of maximal [Ca2+]i
rise (measured using the 50% contour criterion outlined in the
preceding text) evoked by glutamate iontophoresis at a site 251 µm
from the soma was not changed by the addition of AP-5. In the second
cell, the proximal and distal extent of a plateau evoked by glutamate
iontophoresis at a site 271 µm from the soma was decreased 10% by
the addition of AP-5. In neither of these cells did the addition of
AP-5 decrease the rise of
[Ca2+]i evoked by a
just-subthreshold iontophoretic current (data not shown).
To confirm that Ca2+ influx through NMDA channels was not a major influence on the measured rise in [Ca2+]i and to additionally test whether voltage-gated Na+ channel activity might influence the initiation site or spatial extent of the plateau, plateaus were evoked in an additional 10 cells bathed in 100 µM AP-5 plus 1 µM TTX. Figure 5 illustrates typical results when both Na+ channels and NMDA receptors were blocked. Plateaus were evoked in each cell tested under these conditions, and the spatial-temporal pattern of the rise of [Ca2+]i and the spatial extent of the plateaus were similar to those obtained in physiological saline. In these experiments, the estimated proximal extent of the plateaus ranged from 15 to 88 µm (mean: 53.1 µm) and the distal extent ranged from 22 to 146 µm (mean: 49.5 µm). These values were not significantly different from those obtained in physiological saline (for proximal extent P = 0.46; for distal extent P = 0.94; 2-tailed Student's t-test). As found for physiological saline, the proximal extent of plateaus evoked in AP-5 plus TTX was not significantly different from the distal extent (P = 0.81, 2-tailed Student's t-test). Data obtained from plateaus evoked in AP-5 plus TTX are plotted as triangles in Fig. 4 for comparison with plateaus evoked in physiological saline.
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To verify directly that increases in dendritic [Ca2+]i result from Ca2+ influx through voltage-gated Ca2+ channels, iontophoretically evoked changes in dendritic [Ca2+]i were compared before and after the addition of the Ca2+ channel blocker Cd2+ in three cells. Typical results are shown in Fig. 5. The iontophoretic current that evoked the plateau and rise of [Ca2+]i shown in Fig. 5A evoked only a passive membrane potential response and little or no increase in [Ca2+]i in the presence of 200 µM Cd2+ (Fig. 5B). In this and each cell tested, a subthreshold iontophoresis also evoked a significant rise of [Ca2+]i that also was blocked by Cd2+ (data not shown). Altogether, the experiments with AP-5 and particularly with Cd2+ suggest that the measured rise in [Ca2+]i, whether occurring during the plateau or during a subthreshold response, results predominantly from Ca2+ influx through voltage-gated Ca2+ channels. In addition, the results in TTX suggest that neither the site of initiation nor the spatial extent of the plateau depended significantly on Na+ channel activity.
Location and properties of evoked [Ca2+]i increase in basal dendrites
Plateaus could also be evoked by focal glutamate iontophoresis on
basal dendrites (Oakley et al. 2001). In the present
study, the spatial extent of these plateaus was examined in two cells. Figure 6 shows the results obtained in
one of these cells. Focal glutamate iontophoresis at a site 128 µm
from the soma on a basal dendrite evoked an all- (red trace,
bottom)-or-none (gray trace, bottom) plateau. The
corresponding pseudocolor plot (Fig. 6, top) shows that the
plateau-evoked rise of
[Ca2+]i was restricted to
a small region surrounding the iontophoretic site (indicated by dashed
line in pseudocolor plot). A 50% contour drawn around the region
estimates the proximal extent of the plateau as 16 µm and the distal
extent as 15 µm. In this cell, the spatial extent of the plateau was
not significantly changed when 100 µM AP-5 was added to the bath and
the experiment was repeated (data not shown). In the second cell
tested, iontophoresis at a site 105 µm from the soma evoked a
plateau, which was estimated to extend 20 µm proximally and 10 µm
distally.
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DISCUSSION |
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Focal iontophoresis of glutamate on dendrites of neocortical layer 5 pyramidal neurons evoked all-or-none CDRPs, the transient Ca2+ spike and the plateau, which were associated with a maximal rise of dendritic [Ca2+]i around the iontophoretic site. The rise of [Ca2+]i associated both with the CDRPs and with subthreshold iontophoretic responses was blocked by Cd2+ but was insensitive to AP-5, suggesting that Ca2+ influx through voltage-gated channels caused the rise of [Ca2+]i. Since the peak rise of [Ca2+]i always occurred around the iontophoretic site at all locations tested (from 178 to 648 µm on the apical dendrite; cf. Fig. 4), it is unlikely that the plateaus were initiated at discrete "hot spots" on the dendrite. These results strongly suggest that CDRPs can be initiated at any point on the main apical trunk where the membrane potential reaches plateau threshold.
The spatial-temporal pattern of [Ca2+]i rise was used to assess the site of active CDRP initiation, which appeared to extend only over a distance of some tens of micrometers. The decline of [Ca2+]i with distance from the ionotophoretic site strongly suggests that CDRP amplitude also declined with distance (thereby activating fewer Ca2+ channels to result in less Ca2+ influx), and the CDRP was thus propagated decrementally both distal and proximal to its site of initiation.
Most of our data were obtained from iontophoresis on the apical
dendrite. In a previous study (Oakley et al. 2001) we
found that plateaus with electrical properties similar to those evoked on the apical dendrite could be evoked on basal and apical oblique dendrites. In the present study, we measured the plateau-associated rise of [Ca2+]i in the
basal dendrites of only two cells simply to examine whether its
spatial-temporal properties were similar to those observed in the
apical dendrite. The increase in
[Ca2+]i in the basal
dendrites did not extend as far proximally or distally as on the apical
dendrite. This is probably explained by a greater electrotonic decay of
current in basal dendrites than in apical dendrites. In support of this
explanation, we found that the amplitude of basal dendrite plateaus
(measured at the soma) was less than expected for a plateau evoked on
the apical dendrite at a comparable distance from the soma
(Oakley et al. 2001
). Assuming equal plateau amplitudes
at the dendritic site of initiation in both basal and apical dendrites,
a greater decay of the potential with distance in basal dendrites would
result in a smaller plateau amplitude at the soma. The greater decay of
potential with distance in a basal dendrite would also result in less
Ca2+ channel activation away from the initiation
site and thus a more spatially restricted
[Ca2+]i transient. A
recent study that used a different method to apply exogenous glutamate
to the dendritic membrane also found that CDRPs and an associated rise
of [Ca2+]i could be
evoked in basal dendrites (Schiller et al. 2000
). The
spatial extent of the
[Ca2+]i rise measured in
that study was very similar to our values. However, both the rise of
[Ca2+]i and the dendritic
spikes were attributed to current through NMDA receptor channels rather
than voltage-gated Ca2+ channels in that study.
We examined this question in only one of the two basal dendrites that
we examined in this study, and we found both the plateau and the rise
of [Ca2+]i to be AP-5 resistant.
In the present study, most tests of whether Ca2+
influx through NMDA-sensitive glutamate channels contributed to the
rise of [Ca2+]i were
performed on the apical dendrite. We used 100 µM AP-5 to block the
NMDA-sensitive glutamate receptors. This concentration of AP-5 was
chosen because its bath application was found to block the action of
NMDA itself in layer 5 neurons using iontophoretic currents similar to
those employed here (Flatman et al. 1986). There are two
basic questions concerning Ca2+ influx through
NMDA-sensitive glutamate channels. One question is whether NMDA
currents are primarily responsible for the plateau itself. This
possibility is raised by the fact that the cation current through NMDA
channels can be voltage dependent and cause regenerative
depolarizations in the presence of Mg2+
(Flatman et al. 1986
). As discussed in Oakley et
al. (2001)
, we found that Cd2+application
blocked the plateau in every cell tested, and we concluded that
Ca2+ currents through voltage-gated channels were
essential for plateau initiation in most cases. Nevertheless, we also
found that TTX or AP-5 application also blocked or altered the plateau
in a minority of the cells in which these agents were tested,
indicating that inward ionic currents other than
Ca2+ can contribute to the plateau and that the
contribution of these other currents can be essential for the
regenerative response in some cases.
In the context of the present experiments, the important question is
whether the rise of
[Ca2+]i that we measured
is caused by Ca2+ influx through NMDA channels.
The findings of Oakley et al. (2001), that the addition
of 100 µM AP-5 did abolish or alter plateaus in some cells, suggest
that 100 µm AP-5 is sufficient to prevent iontophoresed glutamate
from occupying NMDA receptors at those sites where the receptors play a
significant role in the observed response. No blockade of the rise in
[Ca2+]i by AP-5 was
observed in the present study, but we did not examine the effect of
AP-5 on every recorded cell. We examined the rise of
[Ca2+]i before and after
the addition of AP-5 in two cells and in the presence of AP-5 plus TTX
in 10 others. The measured rise of
[Ca2+]i and its
spatial-temporal pattern in these cells was not significantly different
from those cells not tested with AP-5. Furthermore, Cd2+ completely blocked the rise of
[Ca2+]i (Fig.
5B). Thus we feel confident that the hypothesis that Ca2+ influx through NMDA channels significantly
influenced our results was adequately tested and rejected. Similar
results indicating a minimal role for Ca2+ influx
through NMDA channels and a dominant role for
Ca2+ influx through voltage-gated channels in the
rise of [Ca2+]i that
accompanied subthreshold, electrically evoked, dendritic excitatory
postsynaptic potentials (EPSPs) were obtained in both hippocampal
(Magee et al. 1995
) and layer 5 pyramidal neurons (Markram and Sakmann 1994
).
Recent experiments in hippocampal CA1 pyramidal neurons
(Nakamura et al. 2000) found that the
stimulation of metabotropic glutamate receptors by EPSPs coincident
with influx of Ca2+ through voltage-gated
channels (during action potentials) caused an IP3-dependent release of
Ca2+ from internal stores in the apical dendrites
of those neurons. The spatial extent of the rise of
[Ca2+]i in a CA1 neuron
dendrite was similar to the extent of the 50%-of-peak contour line
that we used to define the active plateau region in our study. Could
the rise of [Ca2+]i that
we observed be caused by release from stores instead of from
Ca2+ influx? Several of our observations suggest
this is not likely to be the case. The application of specific
metabotropic agonists to layer 5 neurons did not evoke plateaus
(Greene et al. 1994
; Linton et al. 1999
).
The metabotropic glutamate receptor (mGluR)-dependent release from
stores occurred in an all-or-none manner in some CA1 cells during
subthreshold EPSPs (without concomitant action potentials),
whereas the rise of
[Ca2+]i was graded
with subthreshold depolarization in our experiments (Fig.
3B) and was abolished by Cd2+ (Fig.
5). If it is accepted that the rise in
[Ca2+]i accompanying
subthreshold, glutamate-evoked depolarizations up to plateau threshold
is mainly due to Ca2+ influx through
voltage-gated channels (because of its
Cd2+sensitivity), then it is difficult to accept
the idea that the [Ca2+]i
rise associated with the very next data point, where the plateau is
triggered (cf. Fig. 3B), is due instead to
Ca2+ release from stores. The latter hypothesis
would be plausible only if mGluR-dependent release from stores were the
cause of the plateau, but as mentioned in the preceding text, specific metabotropic agonists do not evoke plateaus in the layer 5 neurons. Finally, the conditions that caused the greatest release from stores in
the hippocampal neurons (glutamate dependent depolarization concomitant
with Na+ spikes) resulted in a much smaller
rise of [Ca2+]i
and a different spatial-temporal pattern in our experiments than when
the glutamate depolarization triggered a plateau in the absence of
Na+ spikes (our unpublished observations).
Although this question needs to be examined directly in the cortical
neurons, the best explanation for the rise in
[Ca2+]i in our
experiments based on the available evidence is influx through
voltage-gated channels, in which case the rise in
[Ca2+]i reflects plateau amplitude.
We have interpreted the decline of
[Ca2+]i proximal and
distal to the iontophoretic site as reflecting decremental conduction of the plateau away from its site of initiation in both directions. We
use the term "decremental" to express our uncertainty of whether conduction away from the initiation site is purely passive or, as seems
more likely from the observed rise of
[Ca2+]i, that the plateau
is large enough to activate a nonregenerative Ca2+ current that may augment its conduction
compared with the purely passive case. Both the spatially restricted
site of plateau initiation and its decremental conduction toward the
soma suggested by the imaging data are fully consistent with our
previous observations that CDRP amplitude (as recorded in the soma)
declines with the distance of the iontophoretic site from the soma
(Oakley et al. 2001; Schwindt and Crill
1997
). In contrast, an amplitude of the CDRP (measured in the
soma) that was independent of iontophoretic position would be expected
to result from active conduction to the soma. The decremental
somatopedal conduction of a transient Ca2+ spike
evoked in the distal dendrites has been observed directly during
simultaneous intradendritic and intrasomatic recording (Larkum
et al. 1999
). Our present data suggest that conduction toward
the distal dendritic tree is also decremental. Apparently, neither the
initial Ca2+ spike nor the plateau backpropagates
like a Na+ spike.
It is unlikely that this local initiation and decremental conduction of
CDRPs is caused by a significant decline in the density of
Ca2+ channels over the 200-300 µm of the
dendrite that we visualized. Other imaging experiments have suggested
that Ca2+ channels are present along the entire
apical dendritic tree (Markram and Sakmann 1994;
Markram et al. 1995
; Schiller et al.
1995
), and we found that Ca2+ channel
density was sufficient to trigger a Ca2+ spike
and plateau along the entire length of apical dendrite that we
investigated (178-648 µm from the soma).
The absence of an abrupt spatial cutoff of the
[Ca2+]i transient
prevented a precise identification of the region of regenerative Ca2+ influx. A contour line was drawn around the
region where F/F was
50% of the peak
F/F measured during the plateau to estimate the extent of the region of active (regenerative) plateau initiation. Using this criterion, the extent of the active plateau generating region was remarkably small. Even if we were to assume the active region consisted of the entire distance over which fluorescence during
the plateau was significantly higher than fluorescence during a
subthreshold response, this would only amount to a couple hundred
micrometers. Such a distance is still remarkably short considering the
experimental evidence that layer 5 pyramidal neurons are electrically
compact (Larkman et al. 1992
; Stafstrom et al. 1984
). However, the evidence for a long dendritic space
constant was obtained under conditions that minimized the activation of voltage-gated conductances. The activation of K+
currents in particular would tend to decrease the effective space constant and thereby help localize a regenerative event (Wilson 1995
). The conductance increase caused by the opening of
dendritic glutamate channels would further shorten the effective space
constant in the region of plateau initiation. The inability of the
transient Ca2+ spikes to propagate actively was
shown to depend on TEA-sensitive dendritic K+
channels (Schwindt and Crill 1997
), and we suppose the
same is true for the plateau. The importance of dendritic
K+ channels in limiting dendritic excitability
was indicated in the present study by the appearance of
large-amplitude, repetitive, propagating, Ca2+
spikes in response to an iontophoresis that caused only a low-amplitude plateau before TEA application (our unpublished observations).
If Ca2+ spikes actively propagated to the soma in
physiological saline as they do in the presence of TEA, they would
dominate the cell's output because they are of such large amplitude.
Our present imaging data, together with the electrophysiological
results referenced in the preceding text, suggest that the regenerative Ca2+ spike or plateau is normally restricted to a
rather small region of the dendrite and is propagated decrementally to
the soma to cause a much smaller depolarization. In contrast to the
actively propagated Ca2+ spike, the depolarizing
current that reaches the soma from a spatially restricted
Ca2+ spike can sum with depolarizing currents
from other regions of the cell to depolarize the soma. Thus the cell as
a whole remains integrative. According to this idea, the integration of
synaptic input may occur in two stages. There could be a local
integration of synaptic current at one or more dendritic sites to evoke
a local transient Ca2+ spikes and/or a plateau.
The depolarizing current from these local, dendritic, active responses
could then sum with each other near the soma and with synaptic current
from other regions of the cell to evoke the propagated
Na+ spike that is the output of the cell The
rules for integration of plateau currents evoked simultaneously at
multiple dendritic sites were studied in a separate set of experiments
(Oakley et al. 2001).
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ACKNOWLEDGMENTS |
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We thank G. Hinz for technical assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-16792 and by the Keck Foundation.
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FOOTNOTES |
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Address for reprint requests: W. E. Crill, Dept. of Physiology and Biophysics, Box 357390, University of Washington School of Medicine, Seattle, WA 98195-7290 (E-mail: wecrill{at}u.washington.edu).
Received 21 June 2000; accepted in final form 19 February 2001.
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REFERENCES |
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