1Institute of Physiology, University of Bern, CH-3012 Bern, Switzerland; and 2Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany
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ABSTRACT |
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Berger, Thomas,
Matthew E. Larkum, and
Hans-R. Lüscher.
High Ih Channel Density in the Distal
Apical Dendrite of Layer V Pyramidal Cells Increases Bidirectional
Attenuation of EPSPs.
J. Neurophysiol. 85: 855-868, 2001.
Despite the wealth of recent research on
active signal propagation along the dendrites of layer V neocortical
pyramidal neurons, there is still little known regarding the traffic of
subthreshold synaptic signals. We present a study using three
simultaneous whole cell recordings on the apical dendrites of these
cells in acute rat brain slices to examine the spread and attenuation
of spontaneous excitatory postsynaptic potentials (sEPSPs). Equal current injections at each of a pair of sites separated by ~500 µm
on the apical dendrite resulted in equal voltage transients at the
other site ("reciprocity"), thus disclosing linear behavior of the
neuron. The mean apparent "length constants" of the apical dendrite
were 273 and 446 µm for somatopetal and somatofugal sEPSPs, respectively. Trains of artificial EPSPs did not show temporal summation. Blockade of the hyperpolarization-activated cation current
(Ih) resulted in less attenuation by
17% for somatopetal and by 47% for somatofugal sEPSPs. A pronounced
location-dependent temporal summation of EPSP trains was seen. The
subcellular distribution and biophysical properties of
Ih were studied in cell-attached patches. Within less than ~400 µm of the soma, a low density of ~3 pA/µm2 was found, which increased to ~40
pA/µm2 in the apical distal dendrite.
Ih showed activation and deactivation kinetics with time constants faster than 40 ms and half-maximal activation at 95 mV. These findings suggest that integration of
synaptic input to the apical tuft and the basal dendrites occurs spatially independently. This is due to a high
Ih channel density in the apical tuft
that increases the electrotonic distance between these two compartments
in comparison to a passive dendrite.
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INTRODUCTION |
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Neurons in the CNS integrate
thousands of synaptic inputs along their dendrites and generate action
potentials representing their output as a function of time, quantity,
and frequency. Layer V pyramidal neurons of the neocortex have been
used extensively to study these mechanisms. This is mainly due to their
long thick apical dendrite, which is readily accessible for
electrophysiological studies. The apical dendrite traverses all
cortical layers ending in a tuft in layer I. Input on this tuft must
spread along a cable-like structure for 1 mm before reaching the
low-threshold sodium spike initiation zone at the axon initial segment.
Without active "boosting," the synaptic potential would reach this
spike initiation zone greatly attenuated and would thus be nearly
ineffective in shaping the output of the neuron. In addition to the
sodium spike initiation zone, layer V pyramidal neurons have a second,
high-threshold calcium spike initiation zone in the upper half of the
apical dendrite (Schiller et al. 1997
). This led to the
suggestion that distal inputs are functionally separated from proximal
ones (Yuste et al. 1994
). It is therefore important to
understand how both integration sites are functionally coupled by
suprathreshold as well as subthreshold signals. The sodium spike is not
only the output signal but also propagates back into the apical
dendrite (Stuart and Sakmann 1994
). There it combines
with distal input and the two initiation sites interact in a highly
nonlinear fashion (Larkum et al. 1999a
,b
).
Various dendritic conductances may influence subthreshold events in
neocortical pyramidal neurons (for review, see Johnston et al.
1996; Magee 1999b
; Yuste and Tank
1996
). The most profound effects on small membrane deflections
seem to be due to the hyperpolarization-activated cation current
Ih (Schwindt and Crill
1997
; Stuart and Spruston 1998
), which is active
at resting membrane potential (Pape 1996
). Ih significantly alters the
attenuation and summation of dendritic events in pyramidal neurons
(Magee 1999a
; Williams and Stuart 2000
).
In this study, we were asking the following questions. How much are spontaneous excitatory postsynaptic potentials (sEPSPs) attenuated along the apical dendrite in both the somatofugal and somatopetal direction? Is this influenced by Ih deactivation? Where is this conductance situated and what are its properties? Spontaneous synaptic events were recorded simultaneously using three patch pipettes along the apical dendrite of layer V pyramidal neurons. Attenuation of sEPSPs in both directions under control conditions and in the presence of an Ih blocker was investigated. Thereafter cell-attached recordings revealed a high Ih density in the distal apical dendrite. The main findings are that not only are EPSPs not "boosted" along the apical dendrite but in fact are more attenuated than expected from a passive uniform cable model due to the inhomogeneous distribution of Ih. This high Ih density introduces a large electric load in the distal apical dendrite, resulting in an increase of the electrotonic distance between the two spike initiation zones and thus confining the integration of synaptic inputs to one or the other spike initiation zone while only the crossing of threshold is effectively transmitted to the other initiation zone.
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METHODS |
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Brain slice preparation and cell identification
Three-hundred-micrometer-thick parasagittal slices of the
somatosensory neocortex were prepared from 28- to 34-day-old Wistar rats according to federal and institutional guidelines. This was done
in ice-cold extracellular solution using a vibratome (Pelco Series
1000, Redding, CA). Slices were incubated at 37°C for 30-60 min and
left at room temperature until recording. Layer V pyramidal neurons
from the somatosensory area with a long, thick apical dendrite were
visualized by infrared differential interference contrast (IR-DIC)
videomicroscopy (Stuart et al. 1993) using a Newvicon
camera (C2400, Hamamatsu, Hamamatsu City, Japan) and an infrared filter
(RG9, Schott, Mainz, Germany) mounted on an upright microscope
(Axioskop FS, Zeiss, Oberkochen, Germany). The best recordings
with respect to cell quality and ease of access were obtained from
dendrites that were barely visible.
Current-clamp recordings
Current-clamp whole cell recordings were made from three sites
on the apical dendrite (or alternatively, from 2 sites on the dendrite
and 1 on the soma). The compensation of the series resistance across an
electrode can sometimes be unreliable for recording fast
eventsespecially when recording while simultaneously passing current.
Under these circumstances, it is impossible to know exactly what amount
of resistance compensation to use to determine the true membrane
potential. This problem is avoided entirely by the use of three
electrodes. Thus we are able to measure the true voltage responses at
two electrodes while injecting current with a third electrode. We
recorded from similar positions across all neurons investigated
(n = 13) to make general conclusions. Thus we used two
~200-µm segments starting near the soma; this ensured that the main
bifurcation of the apical dendrite came after the most distal
electrode. The most proximal electrode was located at the soma in five
cells and within 100 µm in the remaining eight. The mean
interelectrode distances were 233 ± 32 and 202 ± 37 (SD) µm for the proximal and distal dendritic segment, respectively. This
gave a mean total distance of 435 ± 60 µm. Three Axoclamp-2B amplifiers (Axon Instruments, Foster City, CA) were used. Bridge balance and capacitance compensation was performed on all three electrodes (borosilicate glass tubing with or without 20% PbO; Hilgenberg, Malsfeld, Germany). The resistance was 5-10 M
for somatic and 10-25 M
for dendritic recording pipettes.
Hyperpolarizing rectangular current pulses were injected consecutively
through the three electrodes, and the resulting voltage deflections
were simultaneously recorded from all three electrodes. All traces shown are the average of 20 sweeps. For the study of sEPSPs, 1 µM
bicuculline methiodide was added to the perfusion solution to block
spontaneous inhibitory postsynaptic potentials. Because the occurrence
of sEPSPs is a rare and irregular event in the slice preparation,
recording periods as long as 30 min were needed to gather a sufficient
number of sEPSPs. After a first recording session, 50 µM
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288) (Harris and Constanti 1995
)
or 5 mM CsCl was added to the bath solution to block the
hyperpolarization-activated cation current
Ih. The current injection experiments
were then repeated and sEPSPs again were recorded. Continuous
recordings were filtered at 3 kHz and digitized off-line at 10 kHz with
12-bit resolution using a ITC16 A/D converter. Spontaneous EPSPs were stored on a DAT recorder (DTR-1802, Biologic, France). All experiments were done at ~34°C.
Voltage-clamp recordings
To study Ih currents, somatic
and dendritic whole cell as well as nucleated-patch voltage-clamp
recordings (pipette resistance = 2-4 M) were made by means of
an Axopatch-200A amplifier (Axon Instruments). To obtain nucleated
patches (Sather et al. 1992
), negative pressure
(100-200 mbar) was applied during the withdrawal of the patch pipette.
In cell-attached recordings (pipette resistance = 2.3-5 M
),
the resting membrane potential (Vm;
65.3 ± 4.9 mV) was measured at the end after rupture of the
cell membrane in the whole cell mode, and recordings with a
Vm more positive than
60 mV were
discarded. An outside-out patch was excised after each recording; this
was essential for multiple cell-attached recordings on the dendrite of
the same cell. Data were filtered at 1 kHz using the internal 4-pole
low-pass Bessel filter of the amplifier. The sampling frequency was
twice the filter frequency. Data were digitized and stored on-line
using Clampex8 (Axon Instruments) connected to a personal computer.
Data were analyzed off-line with Clampfit8. All cell-attached traces
shown represent averages of 5-50 sweeps, while the whole cell data
were not averaged. Leak and capacitance of the cell-attached currents
were subtracted.
Data analysis of the current-clamp data
The steady-state attenuation of the induced voltage responses
were measured during the last 20 ms of the transients produced by long
(400 ms, 200 pA) current pulses. The transients recorded with the
current injection electrodes were not used for the analysis due to
possible series resistance compensation problems.
The following transfer coefficients for the electrode positions
i and j were determined (Carnevale and
Johnston 1982): transfer resistance,
Rij = Vj/Ii;
coupling coefficient, kij = Vj/Vi;
attenuation, Aij = 1/kij.
Linearity was assessed applying the reciprocity test;
Rij = Rji
(Koch 1999) (see Figs. 1, A and B,
and 5A). We took advantage of the use of three electrodes to
precisely calculate the input resistance at the middle electrode using
the property of transitivity following directly from the definitions
given above: Rll = RilRlj/Rij where l corresponds to the location between
location i and j, and
Rll corresponds to the input resistance at
location l. In this way, determination of input resistance
did not depend on series resistance compensation.
Single sEPSPs were fitted with an a-b function (Larkum et al.
1996). Because the decaying phase of sEPSPs always fell below baseline (see Fig. 2), the entire shape of the sEPSP could not be
fitted satisfactorily. We therefore restricted the fits to the rising
phase of the sEPSPs. In this paper, we analyze the peak amplitude
because it was the most reliable parameter. sEPSPs were rarely
superimposed because they occurred at a low frequency. Clearly
superimposed sEPSPs were not considered.
Ih currents in cell-attached patches
The amplitudes of the hyperpolarization-activated current were normalized to a pipette tip surface of 1 µm2. For the evaluation of the kinetic properties of Ih, we used primarily large-amplitude current recordings from the distal dendrites. The time constants of the kinetics were determined by least-squares fit of the rising or the decaying phase, respectively. Activation and deactivation time courses were evaluated using a fitting interval of 100 ms after the onset and end of the current, respectively (Fig. 9). The amplitudes of the tail currents induced after switching back to Vm from different clamp potentials were plotted and fitted with a Boltzmann function to obtain the activation range of the current (Fig. 10). The reversal potential (Eh) was extrapolated from a linearly fitted I-V curve obtained from the amplitude of the currents after jumping to different holding potentials from a fully activated state. Using the Goldman-Hodgkin-Katz equation the K+/Na+ ratio was calculated from Eh.
Pooled data are expressed as means ± SD, and tests for statistical differences used an unpaired t-test (assuming nonequal variance) with a significance level of 0.05 (*) or 0.01 (**).
Chemicals and solutions
Slices were continuously superfused with a physiological extracellular solution containing (in mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 20 glucose, bubbled with 95% O2-5% CO2. Pipette solution for current-clamp recordings contained (in mM) 105 K-gluconate, 30 KCl, 10 HEPES, 4 MgCl2, 2 Na2ATP, and 0.3 Na2-GTP, pH adjusted to 7.3 with KOH. Pipette solution for voltage-clamp whole cell recordings contained (in mM) 110 K-gluconate, 30 KCl, 10 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, and 10 Na2-phosphocreatine, pH adjusted to 7.3 with KOH. Biocytin (0.1-0.5%, wt/vol) was added to the pipette solution to visualize the morphology of the pyramidal neurons. Pipette solution for nucleated patch recordings contained (in mM) 140 KCl, 10 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, and 10 Na2-phosphocreatine, pH adjusted to 7.3 with KOH. Pipette solution for cell-attached recordings contained (in mM) 120 KCl, 20 TEA-Cl, 5 4-aminopyridine, 10 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, and 10 Na2-phosphocreatine, pH adjusted to 7.3 with KOH. To study the reversal potential of the Ih current in the cell-attached configuration, we used a high-Na+ solution containing (in mM) 110 NaCl, 2.5 KCl, 20 TEA-Cl, 5 4-aminopyridine, 10 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, and 10 Na2-phosphocreatine, pH adjusted to 7.3 with NaOH.
ZD7288 was a generous gift of Astra-Zeneca (Macclesfield, UK). Tetrodotoxin (TTX) was bought form Alomone Labs (Jerusalem, Israel) and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX 314) was bought from Tocris Cookson (Bristol, UK). All other drugs and chemicals were from Sigma or Merck. Stock solutions of 300 µM TTX and 50 mM ZD7288 were prepared in bidistilled water. Dilution in the extracellular or in the pipette solution provided the final concentrations given below. QX 314 was added to the pipette solution immediately before use.
Neuron staining and reconstruction
After filling the cell with biocytin, the pipettes were
carefully withdrawn and repositioned close to the original location, and a low-power (×5) bright-field image was taken with the video camera. The position of the electrodes was clearly seen on these images, and the distances between the electrodes could be measured to
within ~5 µm using a micrometer scale. Finally slices were fixed in
5% paraformadelhyde and subsequently processed using an
avidin-horseradish peroxidase reaction (Vector Laboratories, Burlingame, CA) (Hsu et al. 1981) to visualize the cell.
Slices were not dehydrated to minimize shrinkage. After coverslipping in Moviol, the stained cells were photographed and reconstructed using
computer-assisted reconstruction systems (Neurolucida or NTS). Only the
most proximal part of the axon was reconstructed. For 11 of 13 cells
studied in the triple current-clamp experiments the morphology could be obtained.
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RESULTS |
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The results will be presented in the following sequence. First, the membrane potentials at the three recording sites will be summarized. Second, reciprocity test, voltage attenuation, coupling coefficient, and transfer resistance for long hyperpolarizing current pulses will be presented. Third, we will present the coupling coefficients and attenuation properties obtained from sEPSPs. Fourth, we will describe summation properties of the apical dendrite. In all these paragraphs, we will compare the results under control and with block of the hyperpolarization-activated current (Ih). A fifth section summarizes the pharmacological properties of the Ih conductance while in the two closing sections its subcellular localization and its kinetic properties are described.
Membrane potential
The recorded membrane potential immediately after breakthrough was
less negative at the middle and even less negative at the distal
electrode than at the proximal electrode: proximal 66.6 ± 7.1 mV, middle
63.0 ± 5.6 mV, distal
60.7 ± 5.0 mV
(n = 13). After the application of 50 µM ZD7288, a
blocker of the Ih current (Harris and Constanti 1995
), the membrane potential
hyperpolarized significantly more at the distal and middle electrode
than at the proximal electrode (proximal
11.0 ± 1.1 mV, middle
13.6 ± 0.5 mV, distal
17.2 ± 0.7 mV; n = 6; P < 0.01). Thus block of
Ih resulted in a similar
Vm at the three recording sites.
Reciprocity, voltage attenuation, transfer resistance, and coupling coefficient for hyperpolarizing current pulses
A fundamental property of a linear system is reciprocity. If
current is injected at location i (e.g., somewhere in the
dendritic tree) and the resulting voltage is measured at location
j (e.g., at the cell body), we record the same voltage at
location i if the identical current is injected at location
j. This is a sensitive test for linearity and was used
throughout this study. This reciprocity test is illustrated for long,
hyperpolarizing (400 ms, 200 pA) current pulses. Pairs of reciprocal
recordings are superimposed under control conditions (Fig.
1A;
n = 13) and after the application of ZD7288 (n = 6) or CsCl (n = 1; Fig.
1B). The color codes of the voltage traces correspond to the
color codes of the electrodes shown in the inset (the same
color code is used in all illustrations). Under control conditions, the
voltage transients show a prominent sag, indicative of
Ih. This sag disappeared completely
under ZD7288, confirming the presence of an
Ih. The nearly perfect superposition of records from different electrodes suggests that the investigated cells behave linearly for the small voltage range tested around the
resting membrane potential, regardless of whether
Ih channels are blocked or not. Note,
however, that such linear behavior does not imply a homogeneous
conductance distribution.
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Estimating voltage attenuation from the site of current injection to the recording site is usually difficult because of uncertainties with the series resistance compensation of the electrodes. However, with three simultaneous recording electrodes we did not need to rely on the voltage transients recorded with the current injecting electrode for determining voltage attenuation, transfer resistance, and coupling coefficients. However, the number of coupling coefficients that can be determined is limited to two, namely to k21 (coupling coefficient from the middle to the proximal electrode with current injected into the distal electrode), and k23 (opposite direction).
Three pairs of voltage transients are shown under control conditions
(Fig. 1C) as well as with ZD7288 (Fig. 1D).
Current was injected into one electrode while the simultaneously
recorded voltage transients from the two other electrodes are
displayed. Such recordings were obtained from all 13 cells under
control conditions and in seven experiments with
Ih block. The pooled data are
illustrated in Fig. 1, E-G. As predicted from the
reciprocity test, steady-state voltage transfer was symmetric
(V13 = V31, 0.8 ± 0.5 mV; i.e., current injected at electrode 1 and voltage measured at electrode 3, and vice versa are identical; true
also for V12 = V21,
1.7 ± 0.7 mV and V23 = V32,
1.3 ± 0.7 mV; Fig. 1E).
Reciprocity was true under control as well as under
Ih blocking conditions
(V13 = V31,
3.4 ± 0.6 mV, V12 = V21,
4.2 ± 0.8 mV, and V23 = V32,
4.8 ± 0.5 mV; Fig. 1E).
The same symmetry was seen for the transfer resistances (Fig.
1F). As expected, mean transfer resistance is lowest from
electrodes 1 to 3 (R13 = R31; 4.4 ± 2.5 M
) due to the long mean
distance between the two electrodes. The mean transfer resistance
between electrodes 1 and 2 was 8.6 ± 3.4 M
(R12 = R21) and
between electrodes 2 and 3 6.7 ± 3.8 M
(R23 = R32). Under
Ih blocking conditions, the values
increased to 17.1 ± 3.2, 20.9 ± 4.0, and 24.0 ± 2.3 M
, respectively. Transfer resistances were used to calculate the
input resistance of the cells at the middle electrode (formula see
METHODS; R22 = 13.3 ± 5.4 M
under control and 29.2 ± 3.5 M
under
Ih blocking conditions). Mean coupling
coefficient k21 was 0.51 ± 0.17 and
k23 was 0.65 ± 0.10 under control and
0.82 ± 0.04 and 0.84 ± 0.08 in ZD7288 or CsCl (Fig.
1G). Thus steady-state voltage amplitude, transfer resistance, and coupling coefficient increased significantly after blocking Ih (Fig. 1, E-G).
Coupling coefficients of sEPSPs
Comparing the sEPSPs recorded simultaneously from the three
electrodes makes it possible to locate the approximate region of origin
for any given synaptic signal (Fig. 2)
(see also Larkum et al. 1998 for the issue of separating
spontaneous events into populations representing synaptic inputs onto
different segments). Identification of the origin of the sEPSPs was
established on the following criteria, which are based on a priori
knowledge of cable properties (Rall 1967
): for sEPSPs
originating proximal to the proximal electrode (e.g., soma and/or basal
dendrites), the amplitude attenuates, while time to peak (ttp) and
latency of the sEPSPs increase from electrodes 1 to
2 to 3 (Fig. 2A). The reverse order is
seen when the sEPSPs originate distal to electrode 3 (e.g.,
in the tuft; Fig. 2B). sEPSPs that were largest at the
middle electrode must have originated somewhere between electrodes 1 and 3 (possibly on an oblique
dendrite; Fig. 2C). Distally elicited sEPSPs are on average
significantly larger and faster than sEPSPs elicited at the soma or
basal dendrites (Vproximal = 1.6 ± 0.8 mV, Vdistal = 2.3 ± 1.6 mV; ttpproximal = 4.5 ± 1.9 ms,
ttpdistal = 2.1 ± 0.9 ms; n = 281 for proximal sEPSPs and n = 351 for distal
sEPSPs). We chose only events that had the largest amplitude at one of
the two outer electrodes. Synaptic contacts given off by a single
afferent fiber are not necessarily restricted to a confined region of
the neuron. sEPSPs from widely distributed locations should lead to
unusual latency progression at the three recording locations and more
or less pronounced inflections. Such composite sEPSPs were excluded
from the analysis.
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Once the origin of the sEPSPs was determined, the coupling coefficients could be estimated by relating their amplitudes recorded from different electrodes. Six coupling coefficients could be determined, namely k13, k31, k12, k21, k23, and k32. The peak amplitudes of sEPSPs measured at the distal electrode were plotted versus the amplitudes measured at the proximal electrode for sEPSPs spreading in somatofugal and somatopetal direction (Fig. 3A). Linear regression lines were fitted through the two data sets. The slope of the regression lines corresponds to the mean coupling coefficient (k31) for somatopetal sEPSPs, while the reciprocal value of the slope of the regression line corresponds to the coupling coefficient (k13) for somatofugal sEPSPs. The coupling coefficient from distal to proximal was, as expected, always smaller than in the opposite direction. Thus sEPSPs transmitted from the soma to the tuft attenuate less than sEPSPs transmitted from the tuft to the soma. Since the sEPSPs are recorded simultaneously with three electrodes, separate coupling coefficients can be determined for the proximal and distal segment. These coupling coefficients are similar for sEPSPs spreading in the same direction (Fig. 3B), and they are approximately twice as large as those determined for sEPSP spreading over the full distance (k31, k13).
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In an infinite cable, the logarithm of the signal attenuation is equal
to the electrotonic distance between the two points of observation
(Zador et al. 1995). This means that the logarithms (ln)
of attenuation for different sections of such a cable are additive. We
have used this relationship as an approximation to extract the length
of the apical dendritic segment over which a typical sEPSP attenuates
to 1/e in both the somatofugal and the somatopetal
direction. In Fig. 3C, ln of mean attenuation is plotted
versus dendritic distance for 10 experiments. Six independent attenuation factors could be determined per experiment; three for each
direction. Linear regression lines are fitted through the origin and
the two data sets. The intersections with ln (mean attenuation) = 1 gives the average physical distance in micrometers over which a sEPSP
with an average time course attenuates to 1/e spreading from
dendrite to soma (273 µm) and from soma to dendrite (446 µm). Thus
attenuation is more pronounced for an EPSP spreading from the tuft
toward the soma than in the opposite direction. We would like to define
these numbers as the AC correlate of the DC length constant. Because
EPSPs have different time courses, this definition is an approximation
and reflects a mean AC length constant for typical EPSPs. This
approximation seems justified since the attenuation of EPSPs is
independent of rise time for rise times longer than 1-2 ms
(Larkum et al. 1998
).
Effects of Ih block on coupling coefficients and "length constants"
Under control conditions, sEPSPs spreading from dendrite to soma show an undershoot and a crossing over in the falling phase (Fig. 4A). After the application of ZD7288, undershoot and crossing over always disappear (Fig. 4A, bottom). The coupling coefficients increased significantly (P < 0.05) for somatofugal sEPSPs only while there was no significant increase in the opposite direction (n = 5 experiments; Fig. 4, C-E). This suggests that the Ih conductance must be situated on the distal part of the apical dendrite, generating a current load at the tuft.
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Block of the Ih leads to an increase in the "AC length constant" of the dendrite. The mean inter-electrode distance was plotted against the average ln of mean attenuation for all three dendritic segments under control and with Ih block (n = 5 experiments; Fig. 4F). Regression lines through each set of data points were used to estimate the distance over which typical sEPSPs attenuate to 1/e in both directions. The nearly perfect correlation suggests that attenuation of sEPSPs is similar in the proximal and distal segment as well as over the combined section of both segments despite the fact that the distal segments tend to be slightly smaller in diameter than the proximal segments. The mean "length constant" of somatopetal sEPSPs increased by 17% from 249 to 292 µm under Ih block. However, this parameter increased more strongly for somatofugal sEPSPs with ZD7288 (from 472 to 695 µm, or 47% more).
The observation, that the coupling coefficient is not decreasing with
increasing sEPSP amplitude (4 mV measured at the proximal electrode)
suggests the absence of a voltage-dependent "boosting" (Figs. 3 and
4). sEPSPs are in fact strongly attenuated by the presence of an
Ih conductance somewhere in the apical
dendrite. Hence this additional attenuation is more pronounced for
sEPSPs spreading in the somatofugal than in the somatopetal direction.
Summation properties of the apical dendrite
Neocortical cells in vivo often discharge in short bursts of high
frequency (Helmchen et al. 1999; Zhu and Connors
1999
), eliciting a brief train of EPSPs in the postsynaptic
cell. We have simulated this situation by injecting five
double-exponential waveforms at 50-Hz frequency (peak amplitude =500
pA;
on = 0.4 ms;
off = 5 ms) (cf. Magee 1999a
). The reciprocity test for such a waveform resulted in a perfect superposition of the two voltage transients recorded at each reciprocal electrode pair, suggesting linear behavior of the investigated neuron (Fig.
5A). This was true for control
conditions and in the presence of ZD7288. The temporal summation
properties of the cells were studied by injecting the current waveform
into one electrode and recording the resulting voltage transients in
the other two electrodes (Fig. 5B).
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The peak amplitudes of the first compared with the fifth EPSP under control condition showed nearly no temporal summation, independent of location and length of the dendritic segments over which the burst of EPSPs spread (n = 4 cells; Fig. 5, C-E). Summation increased under blocking conditions (Fig. 5, C-E). This increase was proportional to the length of the dendritic segment over which the EPSPs traveled but independent of the direction of EPSP propagation (Fig. 5, D and E). This symmetry in the summation behavior under control conditions and with ZD7288 was expected because the reciprocity test led to perfect superposition of reciprocal voltage transients.
With these experiments, it was clearly shown that an Ih conductance has a high impact on EPSP spread along the apical dendrite of layer V pyramidal cells. To study in detail its biophysical properties and the localization on the somatodendritic compartment of this cell type, voltage-clamp recordings were made.
Somatic whole cell voltage-clamp recordings of Ih
When pyramidal cells were hyperpolarized from a holding potential
(Vhold) of 50 mV to potentials
between
60 and
130 mV, all showed an inward current (termed
hyperpolarization-activated current,
Ih; n = 110; Fig.
6A, control) as expected from
the presence of a sag in all cells studied under current-clamp
conditions (Fig. 1, A and C). The amplitude of
the tail currents after clamping the cell from the hyperpolarizing
pulse commands back to a Vh of
70 mV
still increased even after a pulse to
160 mV indicating an
insufficient space clamp. Therefore no further whole cell recordings were used to study the biophysical properties of
Ih.
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Block of Ih
Bath application of 5 mM CsCl (Fig. 6, A and
B; n = 15) or 100 µM ZD7288
(n = 3) blocked Ih
whole cell currents completely (see also Fain et al.
1978; Harris and Constanti 1995
). In addition, the resting membrane potential hyperpolarized by ~10-15 mV. This suggested a reversal potential of the blocked conductance more depolarized than the resting potential. In contrast, bath application of 1 µM TTX, 200 µM 4-aminopyridine, or 10 mM TEA-Cl did not alter the Ih current at all
(n = 22, not shown). It has been shown that ZD7288
exerts its blocking effect at the intracellular side of Ih channels (Harris and
Constanti 1995
), and therefore we have also used 100 µM
ZD7288 in the pipette solution. It took 12-29 min until the
Ih was completely blocked
(n = 3), pointing to a localization of the
Ih channels remote from the somatic
drug injection (Fig. 6C). A blocking effect was also found
by intracellular application of 10 mM QX 314 (n = 3;
not shown), a substance primarily used to block voltage-gated sodium
channels but that has been shown to block
Ih (Perkins and Wong
1995
). To prevent a possible confusion with inwardly rectifying
potassium conductances (Kir)
(Takigawa and Alzheimer 1999
), 200 µM
BaCl2 was applied to the bath. This resulted in a
depolarization of the resting membrane potential, leading to fast
trains of inward currents underlying spontaneous burst firing.
Ih was not affected at all by
BaCl2 (Fig. 6D). These pharmacological
tools identified the hyperpolarization-activated current found in
neocortical layer V pyramidal cells unequivocally as
Ih.
Dendritic localization of Ih
After the whole cell recording of
Ih currents, a nucleated patch was
excised from nine cells (Sather et al. 1992) (diameter ~10 µm; i.e., the nucleated patch contained ~20% of the total somatic surface). No Ih current could
be detected in any nucleated patch, although it was clearly present in
the previous whole cell measurements (Fig.
7, A and B). In
contrast, large voltage-gated K+ and small
Na+ currents could be seen following
depolarization of the nucleated patches (Fig. 7C) (for
comparison, see Bekkers 2000
; Korngreen and
Sakmann 2000
). Thus the lack of
Ih currents in the nucleated patches
could not be due to an artifact of the recording. When patching the
dendrite of the cell from which the nucleated patch was excised
(
50-100 µm from the soma), Ih
currents were detected again (n = 3; Fig.
7D).
|
These data suggest that Ih is not present or in extremely low densities at the soma of the layer V pyramidal cells. To better characterize the distribution of Ih along the cell, we made cell-attached patches on basal dendrites, the soma, and the apical dendrite up to a distance of 820 µm from the soma using a high-K+ pipette solution. At the end of the cell-attached recording, the patch was ruptured to measure the resting membrane potential. The sum of this value and the voltage commands applied revealed the approximate holding potentials.
Ih currents were activated in cell-attached recordings at the apical dendrite at different distances from the soma and the biocytin-filled cell was reconstructed afterward. Ih current density was low up to a distance of ~400 µm from the soma and increased markedly more distally (Fig. 8A). When the current densities measured in 60 patches from 35 cells were plotted as a function of the distance of the recording site from the soma (Fig. 8B), a consistent pattern of current density was found across all neurons. While we found only a low mean Ih density of ~3 pA/µm2 in the soma, basal dendrites, and apical dendrites up to ~400 µm, there was a nonlinear 13-fold increase in the more distal dendrites. The maximal Ih density was in the range of 40 pA/µm2 reflecting a conductance of ~0.03 S/cm2 (Fig. 8B).
|
Biophysical properties of Ih
Ih currents were
activated by clamping cell-attached patches for 500 ms from
approximately 45 mV to values between
85 and
155 mV. The
activation became faster at more hyperpolarized
Vhold (Fig.
9, A and C), and
time constants
were evaluated for the current rise during the 100 ms after the onset of the pulse (see - - - in Fig. 9A).
They were in the range between 31 ± 9 ms at
85 mV and 13 ± 1 ms at
155 mV (n = 5; Fig.
9C). If Ih was activated with a 500-ms pulse to approximately
125 mV and thereafter
deactivated at different Vhold between
105 and
35 mV, the deactivation became faster with more depolarized
Vhold (Fig. 9B). Time
constants were evaluated for the current decay during a 100-ms interval
after the end of the pulse (see - - - in Fig. 9B). They
were in the range of 37 ± 6 ms at
105 mV and 7 ± 1 ms at
45 mV (n = 4; Fig. 9C).
|
After the end of an activating pulse, a tail current could be detected
(Fig. 10A). The amplitude of
this current measured directly after the end of the pulse (see - - -
in Fig. 10A) was plotted against the
Vhold of the previous pulse command.
These relations from five cells were normalized and fitted with a
Boltzmann function (Fig. 10B). The value for half-maximal
activation and steepness of the Boltzmann fit were evaluated for each
cell (n = 5) and averaged. A half-maximal activation at
94.8 ± 6.4 mV and an e-fold current response per
7.8 ± 1.7 mV voltage change were found.
|
The reversal potential (Eh) of
Ih was estimated from the deactivation
protocol shown in Fig. 9B.
Ih currents in cell-attached recordings had a reversal at 4.5 ± 6.8 mV using a
high-K+ pipette solution (n = 4;
not shown). With a high-Na+ pipette solution or
in whole cell mode, this parameter shifted to its physiological value
of 47.7 ± 3.8 mV (n = 4; not shown). Using the
GHK equation a permeability ratio for
PNa+/PK+ of ~0.4 was calculated.
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DISCUSSION |
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The following main findings were obtained. 1) The perfect reciprocity between spatially separated locations indicates that layer V cells can be treated as a linear system at resting potential and for voltage deflections in the range of spontaneous synaptic events. 2) Attenuation of sEPSPs along this apical dendrite is considerably larger than expected for a passive cable with standard values of membrane resistivity and uniform conductance distribution, particularly for somatofugal sEPSPs. This is due to the presence of a hyperpolarization-activated current (Ih). 3) Furthermore this conductance tends to prevent the summation of EPSP-like events irrespective of the location of current injection along the apical dendrite. And 4) the Ih conductance is located preferentially on the distal apical dendrite in a very high density and has relatively fast kinetics.
Layer V pyramidal cell is a linear system in the voltage range of EPSPs
In a linear system, consecutive and reciprocal current injection in a pair of electrodes leads to an identical voltage response in the second electrode (reciprocity rule). This is true for layer V pyramidal neurons at resting membrane potential ±4 mV. This does not, however, imply that the attenuation is the same in both directions since the same current injection results in different voltage responses at the different injection sites. For the voltage deflections studied, the nonlinearity introduced by Ih is not seen in the reciprocity test. This is due to the remote localization of the Ih in the distal dendrite and its hyperpolarized activation range. Therefore the current spread in the proximal dendrite appears to be linear.
By comparing sEPSP propagation at three sites along the apical
dendrite, we were able to examine the homogeneity of dendritic membrane
properties for subthreshold events. The coupling coefficients were
similar for both the proximal and distal segments (Fig. 3B), suggesting that the first half of the apical dendrite (up to ~500 µm) has homogeneous passive membrane properties. However, blocking Ih with ZD7288 resulted in a stronger
hyperpolarization of resting potential at more distal electrodes [this
depolarizing impact of the Ih has also
been described in other cell types (e.g., Holt and Eatock
1995)]. Thus beyond the distal electrode, an increasing Ih density must be expected. In
addition, cable properties predict that boundary effects should play an
ever increasing role as signals approach the distal dendritic tips.
Attenuation for voltage signals spreading along the dendrite is larger
than expected for a passive cable with homogeneous conductance
distribution (see also Schwindt and Crill 1997;
Stuart and Spruston 1998
). This attenuation represents a
measure of the influence that subthreshold signals have on the axonal
site of integration as a function of distance from the soma.
Attenuation is stronger for somatopetal sEPSPs reflected by a smaller
"AC length constant." This difference is due to the large
electrical load that the soma together with the basal dendrites
represents for the approaching EPSP. This difference was predicted for
motoneurons (Rall 1962
) and was recently experimentally
verified in cultured motoneurons (Larkum et al. 1998
).
In contrast, EPSPs at electrotonically remote sites may overcome this
attenuation if they are large enough to activate additional calcium
conductances (Cauller and Connors 1994
; Larkum et
al. 1999b
; Yuste et al. 1994
). Our results
suggest that "boosting" does not occur for small voltage
deflections like EPSPs along the apical dendrite (amplitude
4 mV at
the proximal electrode). EPSP amplification has been reported for large
voltage deflections or under depolarized conditions due to the
activation of a persistent sodium current for depolarizations of
15
mV at the soma (Schwindt and Crill 1995
; Stuart
and Sakmann 1995
; Williams and Stuart 2000
). We
did not measure attenuation in either of these cases.
Summation of EPSPs is prevented by the presence of an Ih conductance
Summation was determined by comparison of the amplitudes of the
first and the fifth EPSP of a train (Fig. 5). We observed that the
summation of a train of EPSP-like waveforms at 50 Hz under control
conditions was negligible and independent on location and distance of
injection and recording electrode. This is true also in CA1 pyramidal
neurons (Cash and Yuste 1998; Magee
1999a
). Application of the Ih
blocker ZD7288 led to location-dependent summation (see also
Williams and Stuart 2000
). If the EPSP train is injected
far away from the recording electrode, summation becomes larger due to
the temporal dispersion of the single EPSP waveform. This is true for
both propagation directions (Fig. 5, D and E). The Ih masks therefore the passive
summation properties of the cell by curtailing the EPSP and generating
an undershoot in its decay time course (see also Williams and
Stuart 2000
) .
Nonuniform distribution of Ih
The attenuation of sEPSPs was markedly reduced when the
Ih conductance was blocked. In
addition, this block led to the disappearance of the undershoot and
crossing over of distally originating sEPSPs. Crossing over has been
shown in pyramidal neurons for somatopetal events and models with
nonuniform conductance distributions are expected to give crossing over
(Koch 1999; London et al. 1999
; Magee 1999a
; Nicoll et al. 1993
;
Stuart and Spruston 1998
; Williams and Stuart
2000
). Cell-attached recordings from different compartments of
the neocortical pyramidal cell revealed a high density of the Ih in the apical dendrite more distant
than 400 µm from the soma (
40 pA/µm2 or
0.03 S/cm2). This nonlinear increase resulted in
an extremely uneven Ih distribution,
even more pronounced than found in CA1 pyramidal cells (Magee
1998
). In another study of layer V pyramidal cells, Williams and Stuart (2000)
have described the
Ih channel density up to ~400 µm
from the soma. They missed the dramatic increase beyond this point that
was revealed in this study because we extended our recordings
820
µm from the soma. The soma itself, the basal dendrites and the
proximal apical dendrite were practically devoid of the
Ih conductance. A similar distribution was also
found in an immunohistochemical study of HCN-1 (Santoro et al.
1997
), one of the four cloned
Ih subunits.
Kinetics and modulation of the Ih
The kinetics of activation and deactivation of the
Ih in our study can be well described
by a monoexponential function with a of ~30 ms at half-maximal
activation. The kinetic data found in this study are nearly identical
to those found by Williams and Stuart (2000)
in another
study on Ih currents in cell-attached recordings from layer V pyramidal cells. In addition, some cells showed
the presence of a second slow exponential function of lower weight.
There is a large variability in the published data for the
Ih kinetics in different native cell
types (see Santoro and Tibbs 1999
). Nevertheless all
data can be roughly classified according to the speed of activation.
One group of Ih channels with time constants in the range of hundreds of milliseconds is present in heart
and thalamus, presumably reflecting the presence of the slowly gating
HCN-2 or HCN-4 subunits of the Ih
channels (Seifert et al. 1999
). In contrast, the time
constants of the other group are much faster (
< 100 ms). They
are found in various central neurons outside the thalamus and may
reflect the presence of fast HCN-1 subunits (Ludwig et al.
1998
; Santoro et al. 1998
, 2000
). In pyramidal
cells of layer V as well as of the CA1 hippocampus, the presence of the
HCN-1 subunit has been shown by in situ hybridization (Mossmang
et al. 1999
; Santoro et al. 2000
),
immunocytochemistry (Santoro et al. 1997
), and
single-cell PCR (Franz et al. 2000
). In addition, the
half-maximal activation at
95 mV is comparable to the data from HCN-1
transcripts. Thus the subunits underlying the
Ih conductance studied here are likely
to be coded by the HCN-1 gene.
It is well known that Ih in heart and
thalamus can be modulated by various G-protein-coupled events
(Di Francesco and Tortora 1991; Lüthi and
McCormick 1998
). This ability seems to be due to the underlying
HCN-4 subunits which are highly cyclic nucleotide sensitive while HCN-1
subunits are not (see Santoro and Tibbs 1999
). Therefore
we would not expect a large effect of cyclic nucleotides on the
Ih in layer V pyramidal cells due to
its molecular constituents.
Functional implications
One effect of a distal region with a high density of
Ih is to isolate the two spike
initiation zones from each other with respect to the inputs integrated.
Back-propagating action potentials are larger than expected for passive
dendrites due to the active boosting by Na+
channels. On the other hand, due to
Ih, EPSPs transmitted in both
directions are smaller than expected from a passive uniform cable
model. This effect is especially marked for high-frequency synaptic
input. Thus subthreshold events have a disproportionately low influence
on the remote spike initiation zone, whereas suprathreshold events have
a disproportionately high influence. The high
Ih density in the distal apical
dendrite introduces a large electric load comparable to that of the
soma resulting in an increase of the electrotonic distance between the
two spike initiation zones. This may therefore be an effective means of
confining the integration of synaptic inputs to one or the other of the
two spike initiation zones while only the crossing of threshold is
effectively transmitted to the other initiation zone. In addition, a
distal region with a high density of
Ih could also prevent large EPSPs from
approaching their reversal potential. A reduced EPSP saturation would
lead to linearization of the relationship between presynaptic input frequency and postsynaptic response (Bernander et al.
1994) and therefore a larger dynamic range.
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ACKNOWLEDGMENTS |
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We thank K. de Peyer for excellent technical assistance and Drs. J. Bischofberger, H.-P. Clamann, W. Senn, and D. Ulrich for helpful discussions and for carefully reading this manuscript.
This work was supported by the Swiss National Science Foundation (Grant SNF 3100-042055.94 to H.-R. Lüscher) and the Silva Casa Stiftung.
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FOOTNOTES |
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* T. Berger, M. E. Larkum, and H.-R. Lüscher contributed equally to this work.
Address for reprint requests: T. Berger, Institute of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern, Switzerland (E-mail: berger{at}pyl.unibe.ch).
Received 19 July 2000; accepted in final form 16 October 2000.
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REFERENCES |
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