Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra ACT 0200, Australia and Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, D-69120 Heidelberg, Germany
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ABSTRACT |
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Bekkers, John M..
Distribution of Slow AHP Channels on Hippocampal CA1
Pyramidal Neurons.
J. Neurophysiol. 83: 1756-1759, 2000.
This work was designed to localize the
Ca2+-activated K+ channels underlying the slow
afterhyperpolarization (sAHP) in hippocampal CA1 pyramidal cells.
Cell-attached patches on the proximal 100 µm of the apical dendrite
contained K+ channels, but not sAHP channels, activated by
backpropagating action potentials. Amputation of the apical dendrite
~30 µm from the soma, while simultaneously recording the sAHP whole
cell current at the soma, depressed the sAHP amplitude by only ~30%
compared with control. Somatic cell-attached and nucleated patches did not contain sAHP current. Amputation of the axon 20 µm from the soma had little effect on the amplitude of the sAHP recorded in cortical pyramidal cells. By this process of elimination, it is suggested that sAHP channels may be concentrated in the basal dendrites
of CA1 pyramids.
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INTRODUCTION |
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Action potentials in neurons are often followed by
a hyperpolarization that may exhibit several distinct phases. Three of these phases are thought to be mediated by three different kinds of
Ca2+-activated K+ channels:
1) high conductance (BK) channels that contribute to the
rapid phase of action potential repolarization; 2) small
conductance (SK) channels that mediate a faster phase of the
afterhyperpolarization (AHP), which lasts up to several hundred
milliseconds; and 3) channels that underlie a very slow
phase of the AHP (sAHP), which may last for several seconds (Sah
1996).
Both BK and SK channels have been studied directly using single-channel
recording techniques (Blatz and Magleby 1987;
Marrion and Tavalin 1998
; Park 1994
). In
contrast, single sAHP channels have remained elusive. Estimates of
their unitary properties have been provided by noise analysis
(Sah and Isaacson 1995
; Valiante et al.
1997
); in CA1 pyramidal cells these channels have a
single-channel conductance of
5 pS. Thus single sAHP channels should
be resolvable in patch-clamp recordings, provided a region of membrane
is found in which the channels are present at sufficiently high
density. Such a region has been suggested by experiments that used a
voltage clamp "switchoff" technique to study the location of sAHP
currents in CA1 pyramidal cells (Sah and Bekkers 1996
).
This work concluded that sAHP channels are concentrated in the proximal
apical dendrite (< approximately 100 µm from the soma) at a density
of roughly one channel per µm2 of membrane,
assuming a uniform distribution.
The present experiments were designed to resolve individual sAHP channels in the proximal apical dendrites of CA1 pyramidal cells. Several approaches were taken in an effort to map the distribution of these channels. It is concluded that many sAHP channels may be found in the basal dendrites of these neurons.
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METHODS |
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Transverse hippocampal slices (250 µm thick) were prepared
from the brains of 2-3 wk old Wistar rats using standard techniques (e.g., Sah and Bekkers 1996). CA1 pyramidal neurons were
identified and patch electrodes were positioned using infrared
videomicroscopy. Recordings were made with two EPC-7 patch-clamp
amplifiers. Patch electrodes had resistances of 3-6 M
(whole cell)
or 15-20 M
(cell-attached). sAHP currents were evoked by a 200-ms
voltage step to
10 or 0 mV from a holding potential of
50 or
60
mV. The bath solution was composed of (in mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 25 glucose (pH 7.4 when saturated with 95% O2-5%
CO2). A HEPES-buffered version of this solution,
used in cell-attached patch electrodes, substituted 25 mM HEPES for the
NaHCO3 and
NaH2PO4. Internal solution
was composed of (in mM) 115 potassium methylsulfate, 20 KCl, 10 phosphocreatine, 4 MgATP, 0.3 GTP, and 10 HEPES (pH 7.2 adjusted with
KOH). All experiments were done at room temperature (22-25°C).
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RESULTS |
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Dendritic cell-attached patch experiments
The first series of experiments sought to resolve single sAHP
channels in the proximal 100 µm of the apical dendrites of CA1 pyramidal neurons (Fig. 1). A patch
electrode in whole-cell mode at the soma was used to elicit a sAHP
current (Fig. 1A, left). A second patch electrode
in cell-attached mode was placed on the apical dendrite of the same
cell 40-100 µm from the soma (70 µm in Fig. 1A) to look
for single sAHP channels. The dendritic electrode contained
HEPES-buffered external solution and its interior was voltage clamped
at 80 mV. Hence, assuming EK
90 mV and a
single-channel conductance for sAHP channels of 5 pS (obtained from
noise analysis; Sah and Isaacson 1995
), the expected
single-channel current was about 0.6 pA (dashed horizontal lines in
Fig. 1A, right), which is likely to be a lower
limit (Valiante et al. 1997
). No channel activity is
apparent in the patch (Fig. 1A, right). Averages
of 10 episodes did not reveal any mean current in the patch with the
timecourse of the sAHP (Fig. 1A, bottom traces;
n = 6 cells).
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In a slightly different version of this experiment, the somatic
electrode was omitted and an extracellular stimulating electrode was
used to apply a tetanus to the alveus. The antidromic action potentials
generated a sAHP in the cell, which was confirmed by a whole-cell
recording from the dendritic electrode at the end of the experiment. In
these experiments the dendritic electrode contained high-potassium
internal solution and its interior was voltage clamped at 0 mV in
cell-attached mode. Again, no suggestion of sAHP channels was found
(n = 9 cells; not illustrated).
A total of 15 experiments revealed no sign of single sAHP channels in
the proximal apical dendrites of CA1 pyramidal cells. This was not a
result of an inability to resolve channels of any sort; outward
channels (presumably K+ channels) were often seen
after the stimulus with a brief latency (Fig. 1B). These did
not have the slow kinetics expected of sAHP channels and were not
blocked by bath application of 4 µM isoprenaline (not illustrated),
which is known to inhibit sAHP channels (Sah 1996).
Dendrotomy experiments
It is possible that sAHP channels are damaged in some way by a patch electrode or are present at too low a density to be found in at least one of only 15 patches. To circumvent these problems, a different approach was taken (Fig. 2). While recording in whole-cell mode from the soma of a CA1 pyramidal cell, two additional patch pipettes were positioned ~30 µm from the soma, the tip of one pipette just above and the other just below the primary apical dendrite of the same cell (Fig. 2, top). At this point little branching of the dendrite had occurred. By slowly lifting the lower of the two pipettes (over 10 min) it was possible to stretch and finally to break the apical dendrite at the location of these pincer pipettes. This could be done without dislodging the somatic electrode.
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This maneuver reduced the amplitude of sAHP current by 44 ± 7% (mean ± SE, n = 6 cells, averaging over the final 10 min; Fig. 2A, filled symbols). Control measurements in which the dendrite was not cut gave a mean reduction of 13 ± 10% (n = 3 cells; Fig. 2A, open symbols; mean amplitude significantly reduced, P < 0.02, unpaired t-test). These results suggest that dendrotomy reduced the sAHP by ~30% compared with control. Illustrative recordings from one cell before and after dendrotomy are shown in Fig. 2B. The input resistance of the cell was increased by a factor 1.6 ± 0.1 (n = 6; Fig. 2C), as expected if a large part of the dendritic tree has been removed (cf. 1.0 ± 0.03, n = 3, for control experiments). The cut was also confirmed for each cell by re-patching with Lucifer yellow that was included in the internal solution; the cell was brightly fluorescent only to the end of the apical stump (not illustrated).
Somatic experiments
The dendrotomy experiments suggest that the majority (~70%) of sAHP channels may be located on the proximal 30 µm of apical dendrite, on the axon or basal dendrites, or on the soma. The next experiments focused on the soma. Dual whole-cell/cell-attached patch recordings were made from the same soma, the protocol otherwise being the same as that for the dendritic experiments described earlier. As in the case of those experiments, no single sAHP channels were seen under the tip of the cell-attached somatic electrode while a sAHP was being evoked via the whole-cell electrode (n = 10 cells; not illustrated).
Again, this negative result may follow from the low density of sAHP
channels. Thus an attempt was made to assay a larger area of the
somatic membrane for sAHP channels while excluding contributions from
other parts of the cell. This was done by pulling a nucleated outside-out patch while continuously recording the sAHP current (Fig.
3). The sAHP steadily decreased in size
as the patch was pulled and was absent from fully isolated nucleated
patches (Fig. 3, trace 4; n = 5 cells). These patches
had resting potentials similar to those of the intact cell (60 mV)
and fired action potentials.
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Axotomy experiments
An attempt was made to patch clamp or cut off the axon or basal
dendrites to determine whether the sAHP channels are concentrated there, but this was not possible in CA1 pyramids because of the small
size and extensive branching of these processes. However, the axon was
able to be cut in Layer V cortical pyramidal neurons where the axon is
prominent (Stuart et al. 1997). Cutting the axon ~20
µm from the soma had no effect on the sAHP measured at the soma (not
illustrated; n = 3 cortical cells). Whereas this result
is only suggestive, it does raise the possibility that sAHP channels
are not found in the distal axons of CA1 pyramidal cells.
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DISCUSSION |
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Prompted by earlier work suggesting that sAHP channels are
concentrated in the proximal apical dendrites of CA1 pyramidal cells
(Sah and Bekkers 1996), this study sought unsuccessfully to resolve single sAHP channels in this region (Fig. 1). Amputation of
the primary apical dendrite ~30 µm from the soma reduced by only
about 30% the sAHP recorded at the soma, compared with control (Fig.
2), suggesting that many sAHP channels must be located in the soma or
very proximal apical dendrite, the axon, or the basal dendrites.
The soma might be excluded because of the absence of sAHP channels in
cell-attached somatic patches and the finding that a sAHP was not
present in nucleated patches (Fig. 3). However, these experiments were
not conclusive. The channels might have been missed in the
cell-attached patches and, although the nucleated patches contained
calcium currents (unpublished observations), these may not have been
large enough to admit sufficient calcium to activate sAHP channels,
even if they were present. It is also possible that the act of forming
a nucleated patch somehow interfered with the activation of sAHP
channels. Despite these uncertainties, it is likely from earlier work
(Sah and Bekkers 1996) that few sAHP channels are
present on or close to the soma. In those experiments the switchoff
current after a somatic voltage clamp step was very fast for IPSCs
known to have a somatic localization, but was relatively slow for the
sAHP current. This result, which was model-independent and highly
reproducible, is difficult to reconcile with a significant presence of
sAHP channels on the soma or on very proximal dendrites.
An axonal localization of these channels remains possible. The axotomy experiments with Layer V cortical pyramidal cells are only suggestive, but indicate that, if CA1 and cortical pyramids are similar in this regard, the sAHP channels would have to be concentrated in the proximal axon less than ~20 µm from the soma.
Finally, the basal dendrites might harbor the majority of sAHP
channels. This conclusion is compatible with all of the results presented here, as well as those of the switchoff experiments, which
could only establish that the channels were remote from the soma and
provided no information about their apical or basal localization
(Sah and Bekkers 1996). However, this conclusion does
appear to conflict with the results of an experiment that used the
shunting of EPSPs by the sAHP to conclude that these channels were not
present in the basal dendrites (Sah and Bekkers 1996
).
Recent findings on the boosting of EPSPs by the subthreshold activation
of a persistent sodium current (Andreasen and Lambert 1999
) suggest however that the shunting experiment of
Sah and Bekkers (1996)
may need to be reinterpreted.
In conclusion, a number of lines of evidence suggest that the majority of sAHP channels are not located in the distal apical dendrites, in the axon, or on the soma of CA1 pyramidal cells. By this process of elimination, it is suggested that the basal dendrites may be enriched in sAHP channels. Future work would need to confirm this suggestion directly.
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ACKNOWLEDGMENTS |
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I thank Prof. Bert Sakmann for support and enthusiasm and Drs. Pankaj Sah and Matthew Larkum for help with pilot experiments. M. Kaiser provided excellent technical assistance.
This work was supported by travel grants from the Ramaciotti Foundations and the Wellcome Trust and a Research Fellowship from the Alexander von Humboldt Foundation.
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FOOTNOTES |
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Address for reprint requests: Division of Neuroscience, John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia.
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 17 May 1999; accepted in final form 2 December 1999.
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REFERENCES |
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