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articles
Somatic EPSP amplitude is
independent of synapse location in
hippocampal pyramidal neurons
Jeffrey C. Magee1 and Erik P. Cook2
1 Neuroscience Center, Louisiana State University Medical Center, 2020 Gravier St., New Orleans, Louisiana 70112, USA
2 Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77050, USA
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Correspondence should be addressed to J.C.M. (jmagee@lsumc.edu)
Most neurons receive thousands of synaptic inputs onto widely spread dendrites. Because of
dendritic filtering, distant synapses should have less efficacy than proximal ones. To investigate this,
we characterized the amplitude and kinetics of excitatory synaptic input across the apical dendrites
of CA1 pyramidal neurons using dual whole-cell recordings. We found that dendritic EPSP amplitude
increases with distance from the soma, counterbalancing the filtering effects of the dendrites and
reducing the location dependence of somatic EPSP amplitude. Dendritic current injections and a
multi-compartmental computer model demonstrated that dendritic membrane properties have only
a minor role in elevating the local EPSP. Instead a progressive increase in synaptic conductance
seems to be primarily responsible for normalizing the amplitudes of individual inputs.
Like many other neurons in the CNS, hippocampal CA1 pyramidal neurons receive tens of thousands of excitatory synaptic
contacts over several hundred microns of their apical dendritic
arborizations1. Dendritic filtering reduces the amplitude and
slows the kinetics of synaptic input. The amount of filtering
directly depends on the electrotonic distance of the synapse from
the final integration site (in this case, the soma or proximal
axon)2–5. Because of the widespread spatial distribution of synaptic input onto these neurons, individual inputs receive widely
varying amounts of filtering depending on synapse location. This
location-dependent synaptic variability would result in distal synapses having less impact on the firing state of the neuron than
their proximal counterparts. There are, however, many potential
mechanisms available to reduce location-dependent synaptic
variability in central neurons. These include passive and active
postsynaptic membrane properties as well as various pre- and
postsynaptic properties of the synapses themselves.
The dendrites of CA1 pyramidal neurons contain a variety of
voltage-gated ion channels that could counter some of the filtering effects of the arborization (reviewed in ref. 6). However, in
CA1 cells, the main impact of these ion channels on synaptic
input seems to be a reduction in the kinetic distortion rather than
a ‘boosting’ of the distal synaptic amplitude7–9 (but see refs. 10,
11). Neuron morphology can also counter location-dependent
synaptic variability. The changes in input impedance and capacitance that occur in distal dendritic compartments (impedance
increases, whereas capacitance decreases) could act to spatially
normalize EPSP amplitude and kinetics to some degree5,12,13. It
is unlikely, however, that the passive geometry of pyramidal neurons can substantially reduce the location-dependent variability
of relatively high-frequency signals like synaptic inputs.
If dendritic mechanisms for reducing location-dependent
amplitude variability do exist, they are most likely to be found
at the synapse itself. In support of this, the distal apical dendrites
nature neuroscience • volume 3 no 9 • september 2000
of both neocortical and hippocampal pyramidal neurons are
more sensitive to glutamate than the proximal dendrites 14
(A. Frick, W. Zieglägansberger & H.U. Dodt, Soc. Neurosci. Abstr.
24, 325, 1998). This may indicate an increase in local synaptic
amplitude that, with distance, could counterbalance the amplitude-filtering effects of the dendrites. Others have proposed that
an increase in synaptic conductance with distance from the soma
reduces the location dependence of synaptic activity15–18. We set
out to directly examine this idea by determining the location
dependence of AMPA receptor-mediated excitatory synaptic
input in hippocampal CA1 pyramidal neurons.
RESULTS
Using dual whole-cell recordings from hippocampal CA1 pyramidal neurons, we simultaneously measured the amplitude of
putative unitary (single terminal) EPSPs both at the dendritic
input site and at the soma (Fig. 1a and b). The location of the
synaptic input and the dendritic recording pipette were varied
together from cell to cell, which insured a recording directly from
the site of input paired with another at the soma. Unitary events
were evoked primarily using the localized application of high
(600 mOsM) osmolarity external solution to the apical dendrite
around the dendritic recording pipette. Minimal stimulation
techniques were also used (Methods).
For each cell, all EPSPs recorded at the soma were used to
calculate an average somatic EPSP, and all EPSPs simultaneously recorded at the input site were used to calculate an average dendritic EPSP (Fig. 1d). To determine the location
dependence of unitary input, we plotted the amplitudes of the
average somatic and dendritic EPSPs against synapse location
for the entire group of cells (Fig. 1e). The amplitude of the
local dendritic EPSP, recorded at the site of input, increased
nearly fourfold with distance of the synapse from the soma
(0.25 to 0.8 mV from 50 to 325 µm; Fig. 1e). On the other
895
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Fig. 1. Synaptically evoked EPSP amplitude at the
soma is independent of synapse location.
(a) Dendritic membrane potential just before the
localized application of high osmolar external solution (600 mOsM) to a distal dendritic location
(∼300 µm, upper trace, baseline). Application of
high osmolar external solution results in the spontaneous occurrence of EPSPs that are simultaneously recorded at both the dendritic input site (d)
and at the soma (s; lower traces, +sucrose). (b) In
another neuron, EPSPs are evoked more proximally (∼50 µm) and recorded from the dendritic
site (d) and the soma (s). (c) Scatterplot of dendritic and simultaneously recorded somatic EPSP
amplitude from the cell shown in (a).(d) The average of all EPSPs simultaneously recorded both at
the site of input (dendrite) and at the soma for the
neuron receiving distal input (distal; average of 93;
same cell shown in a) and for the neuron receiving
proximal input (prox; average of 122; same cell
shown in b). Dendritic amplitude is largest in the
neuron receiving more distal input, whereas
somatic EPSP amplitude is similar for both neurons. (e) Mean EPSP amplitude for all cells plotted
as a function of input distance from the soma
for both dendritic and somatic recordings.
(f) Cumulative amplitude histograms for the above
recordings showing that the distribution of distal
dendritic EPSPs (light solid line) is skewed to the
right compared to more proximal dendritic EPSPs
(dark solid line). Also the distributions of EPSPs
from both input locations are similar once they
reach the soma (distal, light dashed line; proximal,
dark dashed line). Traces were digitally smoothed
as in Methods. All EPSPs shown were evoked by
high osmolar solution.
a
b
d
c
e
hand, EPSPs recorded simultaneously at the
soma exhibited an average amplitude that
was virtually independent of synapse location (Fig. 1e; ∼0.2 mV from 50 to 325 µm;
EPSC amplitudes ranged from 4.3 to 5.6 pA
for four recordings from 50–290 µm). The values for somatic
EPSP/Cs are similar to those reported for single synaptic or
quantal events recorded at the soma17–23.
Cumulative histograms of EPSP amplitudes were generated
for both local dendritic and propagated somatic EPSPs (Fig. 1f).
The amplitude distribution of distal events recorded at the dendritic site of input was shifted toward larger amplitudes when
compared to proximal events recorded at the input site (Fig. 1f,
light solid line versus dark solid line; range of distal, 0.1 to 4 mV;
proximal, 0.1 to 1.5 mV). Following propagation to the soma,
however, the amplitude distributions of both proximal and distal
inputs were similarly shaped and showed a smaller range of
amplitudes than those recorded at the dendrite (Fig. 1f, dashed
lines). These data suggest that the amplitude of a local dendritic
EPSP is increased sufficiently to counter the electrotonic filtering it will experience as it propagates to the soma. The final result
is that the mean amplitude of all synaptic potentials at the soma
does not depend on synapse location. We next examined the distinct roles of active and passive postsynaptic membrane properties, as well as the properties of the Schaffer collateral synapses
themselves, in producing this effect.
To examine the contribution of postsynaptic membrane
properties, we bypassed the synaptic machinery by injecting
896
Average EPSP
f
Peak amplitude (mV)
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articles
EPSC-shaped currents (30 pA, τr = 0.1 ms, τd = 3 ms) directly
into the dendrites. Local dendritic and propagated somatic
voltage transients (EPSP i ) were simultaneously measured
(Fig. 2a). In contrast to the synaptically generated EPSPs, the
EPSP is were location dependent: somatic EPSP i amplitude
decreased to less than half as the input location was moved distally (0.5 to 0.19 mV from 50 to 325 µm; Fig. 2b). The amplitude of the EPSPi at the dendritic injection site did increase
slightly with distance from the soma (∼50%, Fig. 2b) but much
less than the synaptically evoked EPSP (Fig. 1e). The level of
amplitude attenuation from the site of input to the soma was
similar for synaptically evoked EPSPs and EPSPi (from ∼80% at
the most distal to ∼20% at the most proximal; Fig. 2c), demonstrating that a difference in propagation was not responsible
for the location dependence of the EPSPi. Together these data
indicate that dendritic membrane mechanisms are not sufficient to increase the local dendritic amplitude of the EPSP and
counter dendritic filtering. Therefore, as current injections of
proper size and shape could not reproduce the spatial EPSP
profile observed with actual synaptic input (< 50%; Fig. 1d),
we conclude that the passive structure and active properties of
CA1 dendrites cannot account for the reduction in the location dependence of synaptic input.
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articles
Soma rise
Distance (µm)
(dend – soma)/dend
reported in these cells and is presumably the result of both active
and passive dendritic membrane mechanisms8,9. The duration
of the synaptic conductance is unlikely to depend on location
because there was a similar change in the decay time constant of
both the local EPSP and the EPSPi (Fig. 3b).
Local rise
Soma decay
f
Local decay
Tau (ms)
e
Amplitude (mV)
Distance (µm)
d
Distance (µm from soma)
Distance (µm from soma)
Local decay
g
Local rise
Tau (ms)
Tau (ms)
c
c
b
Amplitude (mV)
h
Local decay
Tau (ms)
Distance (µm)
Prox
Distal
Tau (ms)
b
Tau (ms)
Local rise
Tau (ms)
a
a
Peak amplitude (mV)
The location and amplitude dependence of EPSP kinetics can
reveal the forces shaping EPSP propagation. Therefore both the
actual synaptic events as well as EPSPi were fit by an exponential
function that provided time constants for the rising (τr) and
decaying (τd) phases of the synaptic potentials. We then examined their dependence on synapse location (Fig. 3a–d) or amplitude (Fig. 3e–h). For local (dendritic) events, the rise time of
EPSPs of similar amplitude (0.2–0.6 mV) did not seem to depend
on location (Fig. 3a). On the other hand, both the EPSP and
EPSPi decay time constants decreased substantially with distance
from the soma (Fig. 3b). A similar location dependence has been
Tau (ms)
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Fig. 2. The somatic amplitude of EPSPs generated by uniform current
injections depends on input location. (a) Traces are the average voltage
transients (EPSPI, average of 5 sweeps) recorded in the dendrite (dendrite) and simultaneously at the soma in response to EPSC-shaped current injections into the dendrites. These recordings are from the same
neurons shown in Fig. 1 (distal, Fig. 1a; proximal, Fig. 1b). The dendritic EPSPi amplitude is slightly increased in the more distal dendritic
recording, whereas the somatic EPSPi is much larger for proximal input.
(b) The average EPSPI amplitudes are plotted as a function of input distance from the soma for both dendritic () and somatic () recordings.
Note that dendritic EPSPi amplitude does not increase as much as
synaptic EPSP amplitude (Fig. 1c) and that somatic EPSPI amplitude
decreases with distance. (c) EPSP attenuation is plotted as a function of
input distance from the soma for both synaptic EPSPs () and currentinjection EPSPI (). Attenuation is expressed as the decrease in EPSP
amplitude (dend - soma) relative to the initial amplitude (dend). Traces
were digitally smoothed as described in Methods.
Distance (µm)
Amplitude (mV)
Amplitude (mV)
Fig. 3. Location and amplitude dependence of EPSP kinetics. Rise (a) and decay (b) times for average EPSPs recorded at the dendritic input site as a
function of distance from the soma (time constants from fit by an exponential function; fits are constrained to the first 5–6 ms of the EPSP decay phase
for decay times). There is no location dependence to EPSP rise times, whereas decay times drop sharply with distance. Rise (c) and decay (d) times for
average EPSPs recorded at the soma, as a function of input distance from the soma (propagated EPSPs simultaneously recorded with the dendritic EPSPs
plotted above). Somatic EPSP rise times show a more pronounced location dependence than do somatic EPSP decay times. EPSPs evoked by high osmolar solution (, solid line fits) show similar location dependence as EPSPi produced by dendritic current injections (∆, dashed line fits). (e–h) Rise and
decay times for all EPSPs recorded at a distal (e, f) or proximal (g, h) input site as a function of EPSP amplitude. The rise times of both distal and proximal EPSPs (e, g, , solid line fits) increase with amplitude, whereas the rise times of EPSPi do not (e, g, ∆, dashed line fits). Decay times for distal events
do not show any amplitude dependence (f). Proximal events show a slight increase of decay time as amplitude increased (h). Data points were fit by
either a linear or single exponential function. Kinetics from EPSPs evoked by high osmolar stimulation.
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a
b
100 µm
Fig. 4. An increase in synaptic conductance can account for
the normalization of somatic EPSP amplitude in a passive
computer model. (a) Reconstructed CA1 pyramidal neuron
used in multi-compartmental model. (b) EPSPs recorded at
the site of input (dendrite) and simultaneously at the soma
(soma) for a proximal (proximal; 100 µm from the soma) and
more distal (distal, 300 µm from the soma) synaptic input of
uniform conductance (872 pS). Also shown are EPSPs for the
more distal input with the synaptic conductance increased
approximately twofold (distal X2, 1600 pS). Inset, proximal
and distal dendritic EPSPs with amplitude normalized to
show differences in decay kinetics. (c) EPSP amplitudes plotted as a function of input distance from the soma for both
dendritic () and somatic () recordings for the 872 pS
synaptic input. Note that the dendritic EPSP amplitude is not
sufficient to remove the decrease in somatic EPSP amplitude
with distance. (d) Synaptic conductance required to produce
a location-independent 0.2 mV EPSP at the soma for a variety
of different membrane parameters. (Each line is the result of
a quadratic fit.) Note that an increase in synaptic conductance is required in all conditions. m, specific membrane
resistance Rm; a, axial resistance Ra.
c
d
Peak amplitude (mV)
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articles
The propagated events recorded simultaneously
at the soma showed the expected location-dependent
rise time, with the time constant increasing over
threefold across the range of input locations (Fig. 3c).
The location dependence of the decay time for propagated EPSPs was reduced because the local decrease
in decay time counters the filtering effects of the dendrites to some degree9 (Fig. 3d). Overall, however,
there were no significant differences between the kinetics of the
true synaptic events and the voltage transients induced by current injection. These data indicate that the somatic EPSP rise
time is a more accurate indicator of synapse location than EPSP
duration and further demonstrate the role of dendritic membrane properties in shaping EPSPs.
We next examined the amplitude dependence of EPSP kinetics
for both distal and proximal local dendritic events (Fig. 3e–h). There
was a direct relationship between the EPSP rise time and amplitude, with the rise time increasing nearly threefold for distal and
about twofold for proximal synapses across the range of amplitudes
(slopes, proximal, 109 ± 11 ms/mV; distal, 91 ± 8 ms/mV; Fig. 3e
and g). There was no such observable amplitude-dependent
increase in the rise time of the EPSPi (Fig. 3e and g), suggesting a
synaptic mechanism for the increase in EPSP rise time with
amplitude. There was very little amplitude dependence to the
decay time constants other than a slight increase that was occasionally observed at proximal sites (both EPSP and EPSPi; Fig.
3f and h). As discussed further below, the amplitude-dependent
increase in EPSP rise time suggests a mechanism underlying the
increase of distal EPSP amplitude.
The experimental evidence thus far suggests the mechanism
for eliminating location dependence is centered at the synapse.
We therefore sought to determine if a simple increase in synaptic conductance could theoretically account for this effect, using
a realistic passive multi-compartmental computer model of a
CA1 pyramidal neuron (Fig. 4a; Methods). To determine the
extent of location dependence in the model, we placed synaptic
conductances in the apical dendrites at varying distances from
the soma. The conductance amplitude was adjusted such that
a proximal synapse (located 50 µm from the soma) produced
a somatic EPSP of 0.2 mV (Fig. 4b). As this synaptic conduc898
tance was moved farther into the dendrites, the local dendritic
EPSP amplitude and decay rate increased (Fig. 4c), whereas the
EPSP amplitude at the soma decreased as a function of distance
(Fig. 4c). A progressive increase in the conductance of the
synapses with distance from the soma could remove the location dependence for a realistic range of passive parameters
(Fig. 4d). (An approximately 2-fold increase was required for
a synapse located 300 µm from the soma.) In each case, the
synaptic input produced a 0.2 mV EPSP at the soma (example
in Fig. 4b), regardless of its dendritic location. These simulations, together with the above experimental observations, suggest that an increase in synaptic conductance with distance from
the soma, and not postsynaptic morphology or voltage-dependent mechanisms, account for the elimination of somatic EPSP
location-dependent variability.
If an increase in AMPA receptor-mediated synaptic conductance is indeed the mechanism for eliminating the effect
of distance on the synaptic response, then it should be possible to directly measure this using a dendritic voltage clamp.
We measured EPSCs at the site of synaptic input across the
entire axis of the apical dendrite receiving Schaffer collateral
input using four different techniques for stimulating localized
single synaptic activity: minimal electrical stimulation, local
application of high osmolar external solution (Fig. 5a), low
electrical stimulation in locally applied Sr2+-containing external solution, and local application of adenosine A1 receptor
antagonist. As with the EPSPs, mean EPSC amplitude increased from ∼8 pA for synaptic locations near 100 µm to
∼24 pA for synapses around 300 µm from the soma (Fig. 5b
and c). All four stimulating techniques produced EPSCs of
similar mean amplitude, with EPSCs evoked in Sr2+ having a
slightly lower mean amplitude across the dendritic range. This
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articles
Fig. 5. Dendritic EPSC amplitude increases with synapse distance. (a) Dendritically recorded EPSC activity induced by
local application of high-osmolarity external solution onto a
proximal (∼100 µm; top trace) and a distal (∼280 µm; bottom trace) dendrite of two different neurons. (b) Average of
all EPSCs recorded from the two neurons shown in (a).
(c) Mean EPSC amplitude as a function of input distance from
the soma for EPSCs induced by high osmolarity () or
EPSCs evoked in normal () or Sr2+-bcontaining external
solution (). (d) Normalized EPSC amplitude histograms for
the recordings in (a). The distribution of distal EPSCs (open
histogram) contains many more large-amplitude events, and
a larger mean, than the histogram for proximal EPSCs (filled
histogram). Inset, cumulative histogram (distal, light line;
proximal, dark line). (e) Synaptic charge, normalized to the
value of the proximal synapses, as a function of distance from
the soma. Data were grouped into proximal (75 ± 25 µm),
middle (175 ± 25 µm) and distal (275 ± 25 µm) regions.
Integrated EPSCs were induced by high osmolarity or evoked
in Sr2+-containing external solution. *p < 0.05; **p < 0.02.
a
b
may indicate that there is a relationship between
EPSC amplitude and probability of release, which
should be somewhat lower when Sr2+ is used as the
main divalent 24. Amplitude distributions demon- d
strated that mean EPSC amplitude of distal synapses is increased because of the presence of
larger-amplitude events that are not found in proximal synapses (Fig. 5d). Current–voltage relationships showed that there was no difference in the
reversal potentials, and therefore driving forces,
between the proximal and distal synapses (proximal,
7.3 ± 1.3 mV; distal, 6.0 ± 2.3 mV; n = 3 in both, not
corrected for junction potential).
Because imperfect dendritic voltage-clamp conditions would allow location-dependent filtering to affect
the amplitude and shape of recorded EPSCs (Fig. 2b;
Discussion), we also compared synaptic charge. As synaptic
charge is less affected by filtering, comparing the integral of the
EPSC should provide a relatively filtering-independent assessment of synaptic conductance. The synaptic charge measured
from the EPSC integrals increased more than twofold from proximal to distal Schaffer collateral synapses (Fig. 5e), further indicating an increase in synaptic conductance with distance.
To directly compare the extent of voltage clamp in the distal and proximal dendritic regions, we placed two recording
pipettes within 20 µm of each other on the same dendrite.
EPSC-shaped current injections were delivered via the most
distal electrode and the voltage deflection recorded either with
both pipettes in current-clamp mode or with the more proximal pipette in voltage-clamp mode (Fig. 6a). The level of voltage clamp, quantified as the ratio of the EPSPi integrals, was
similar for both proximal and distal recordings (voltage
clamp/no clamp, 9% proximal, 11% distal; n = 2 for both
groups). Although the clamp was similar, and quite good, for
both proximal and distal recordings, it was nevertheless imperfect. As a result, the amplitude and decay rate of the EPSCi (the
EPSCs resulting from current injections) were slightly location
dependent, with distal events having a somewhat larger amplitude and faster decay rate (Fig. 6a). EPSCi rise time was independent of location.
A similar decrease in decay time constant with distance
from the soma was observed in the synaptic EPSCs (Fig. 6b).
nature neuroscience • volume 3 no 9 • september 2000
c
e
The similarity of the location dependence of the actual EPSC
decay and that of the EPSCi (Fig. 3b) suggest that the faster
decay of the distal events results from dendritic morphology
and an imperfect voltage clamp and not from any kinetic differences in synaptic conductance. A similar conclusion was
reached above for the apparent location-dependent differences
in EPSP decay rates (Fig. 3b).
Although the rise times of similarly sized EPSCs were independent of location (77.8 ± 4.8 µs, n = 9; 79.4 ± 3.9, n = 10, for
∼10 pA proximal and distal EPSCs, respectively), a marked
amplitude dependence of the EPSC rise was noted (Fig. 6c). In
nearly all cells, the rise time constant increased with EPSC
amplitude in an approximately linear fashion (proximal slope,
9.7 ± 1.1 µs/pA, n = 9; distal slope, 6.3 ± 1.4 µs/pA, n = 8). The
difference in slope results from an apparent saturation of the
rise times at larger amplitudes that are only present in the distal recordings (notice exponential fit in Fig. 6c). There was no
amplitude dependence to the EPSCi even for a twofold larger
range of current amplitudes (Fig. 6c, left traces). These data
suggest that the slowing of EPSC rise time with increasing
amplitude is the result of an actual kinetic change in the synaptic conductance and not of voltage-clamp imperfections.
The amplitude distributions of both proximal and distal
EPSP/Cs exhibited a large amount of variability and a prominent peak at smaller amplitudes (Fig. 7a–c). This peak could
be observed when the distributions were fit by a single Gauss899
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Fig. 6. Location and amplitude dependence of EPSC
kinetics. (a) Paired recordings from either proximal (left
traces, Prox) or distal (right traces, Distal) dendritic
regions from different neurons. Large EPSPi (no clamp)
evoked by current injection with both electrodes in current-clamp mode. Smaller EPSPi (clamp) evoked by current injection through one electrode in current-clamp
mode and the other in voltage-clamp mode. EPSCi
recorded by the voltage-clamp electrode (lower traces, I)
in response to the current injection. The EPSPi labeled
clamp is the escape voltage of the dendritic region and is
similar between the regions. (b) The left traces are the
exponential fits of the EPSCis in (a) with their amplitudes
normalized. The middle traces are exponential fits of
synaptically evoked EPSCs (from recordings in Fig. 5).
Note that the distal EPSCs always decay faster than the
proximal even with the EPSCi that were evoked by a uniform current injection. Plot is decay time constant versus
input location for synaptically evoked EPSCs () and current-injection EPSCi (), showing that the decay times of
both events decrease similarly with distance. (c) Left
traces, EPSCi (same recordings as in a) for a range of current injection amplitudes, showing that rise time remains
constant. Middle traces, selective averages of synaptically
evoked EPSCs (from another dendrite ∼290 µm). EPSCs
with amplitudes 3–7 pA, 8–12 pA, 13–17 pA and 18–22 pA
were averaged, respectively. Note that the rise time of
these events slows as the amplitude increases. Plot is rise
time constant verses EPSC amplitude for all synaptically
evoked EPSCs (from same dendrite), showing that the
rise times slow with EPSC amplitude. Number of points
in the traces in (c) have been reduced to one third for
clarity. Traces are digitally smoothed. All EPSCs shown
were evoked by high osmolarity.
a
b
c
ian function that was constrained to the smaller amplitudes
(0–9 pA range) or as the first of several peaks when fit by a
multi-Gaussian function. The mean of this first peak was similar across the entire range of dendritic synapse locations
(5–6 pA and 0.15–0.20 mV; < 50% increase from 50 to 300 µm;
Fig. 7d). These data suggest that there may be an increase in the
number of quanta released (quantal content) from synapses
with distance from the soma. However, the coefficient of variation (CV), which should directly reflect quantal content, does
not increase with distance (Fig. 7e). Instead there is a greater
than twofold increase in the variance divided by the mean
(σ2/x), which suggests that mean quantal size, not quantal content, increases with distance (Fig. 7e). Together these data imply
that there is a population of larger-amplitude, slower-rising,
unitary events increasingly present at more distal Schaffer collateral synapses.
Following propagation to the soma, the amplitude of the
smallest synaptic EPSPs and EPSP is showed a pronounced
dependence on input location (open circles; Fig. 7d). For input
located more distal than 125–150 µm, the amplitude of the smallest EPSPs at the soma was lower than the noise for single sweeps
(< 0.05 mV) and would normally have been missed (counted as
failure) if not for the dendritic recording (Fig. 7c). Beyond about
200 µm, the events seem to lose all ability to significantly change
the voltage at the soma, even in selected averages. This demonstrates the difficulties of using only somatic recordings to characterize synaptic events and indicates that the use of such
recordings to determine failure rates or the presence of silent
synapses should be limited to only the most proximal locations
(within 150 µm of the soma).
900
Synaptic EPSC
Synaptic EPSC
Decay tau (ms)
EPSC rise tau
DISCUSSION
We have characterized the amplitude and kinetic properties of
AMPA receptor-mediated synaptic input across a wide range of
the apical dendrites in CA1 pyramidal neurons. The main finding is that the mean amplitude of the synaptic conductance
increases with distance from the soma and that this increase
counterbalances dendritic filtering, dramatically reducing the
location dependence of synaptic amplitude. The use of realistic
dendritic current injections and a passive multicompartmental
computer model demonstrate that the passive and, most likely,
the active properties of CA1 dendrites have only a minor role in
elevating the local EPSP amplitude of distal unitary inputs. Voltage-gated ion channels, however, significantly shape the amplitude of larger EPSPs 6,7,10,11. Instead of dendritic membrane
properties elevating the amplitude of distal dendritic EPSPs, it
seems that a progressive increase in synaptic conductance, with
distance from the soma, is primarily responsible. Both in the hippocampus and in other CNS regions, location dependence of
synaptic efficacy is minimal, with several authors suggesting an
increase in synaptic conductance as a mechanism15–18,24,25. Our
data, recorded directly from the site of input, fit well with these
other studies.
What is the mechanism of the increased synaptic conductance
at distal synapses? There are several possibilities: increases in
AMPA receptor number or density, agonist affinity, single-channel conductance, quantal glutamate concentration, or number
of quanta released per terminal. Increases in cleft glutamate or
AMPA receptor density, affinity or single-channel conductance
seem unlikely to be responsible. In all of these cases, we should
see an increase in the amplitude of the smallest events with disnature neuroscience • volume 3 no 9 • september 2000
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tance from the soma, and rise times should not increase with
amplitude22,25,27. Therefore, it seems most likely that the increase
in distal synaptic conductance results from an increased number
of AMPA receptors and/or quanta released.
Terminals capable of releasing multiple quanta from multiple
release sites are observed in several cell types, including Schaffer-collateral synapses of hippocampal CA1 pyramidal neurons 22,23,25,28–30. Theoretically, the number of multisynaptic
boutons or the number of release sites per bouton could increase
with distance from the soma. Also, Schaffer collateral synapses
have a range of areas, and the number of AMPA receptors at
these synapses is directly related to the area31,32. Thus it is possible that quantal release at a larger synapse would open more
AMPA receptors at lower cleft glutamate concentrations. Such
a situation could produce larger-amplitude currents with a slower rise time33. Either of these two mechanisms should correlate
with an elevated dendritic glutamate sensitivity 14 (A. Frick,
W. Zieglägansberger & H.U. Dodt, Soc. Neurosci. Abstr. 24, 325,
1998). Given the complexity of these synapses, however, a thorough determination of the exact mechanisms involved in the
increase in distal synaptic conductance as well as the processes
involved in producing and maintaining this gradient will require
more direct experimentation.
Several lines of data now indicate that, contrary to theoretical expectations, there is minimal location dependence to multiple components of dendritic integration, including unitary EPSP
amplitude and temporal summation. Also, the threshold for
action potential generation increases with distance from the soma
at a rate that is very similar to that of the increase in local synaptic amplitude6,7. Because of this, all synaptic input should be
equally distant from threshold no matter where it is in the cell.
In this situation, all synapses will have the same ability to initiate action potentials and to induce long-term synaptic plasticity
regardless of their location in the dendritic arborization6. This
elimination of location from the synaptic weight is critical for
Hebbian-type processes, where the weight of any given synapse at
the final integration site primarily depends on its history of use
and not on other factors such as synapse location34. Furthermore,
normalization of synaptic input can reduce the variability of
action potential firing in single neurons and improve the synchrony of neuronal population activity9.
CA1 pyramidal neurons have equalized the impact of synaptic input while preserving the location independence of synaptic integration and plasticity through a beautifully intricate
interplay between dendritic membrane excitability and singlesynapse properties. However, this is only half the story, as all
the ion channels responsible for this balanced interplay are highnature neuroscience • volume 3 no 9 • september 2000
Count
Proximal
peak 1 = 4.7 pA
b
Count
a
EPSP
peak 1 = 5.8 pA
EPSC amplitude (pA)
EPSC amplitude (pA)
c
Distal
d
Small event amplitude
Count
Fig. 7. Amplitude of smallest dendritic events shows little dependence
on synapse location. Amplitude distributions for dendritically recorded
EPSCs from proximal (a, ∼50 µm) or distal (b, ∼300 µm) locations.
Distributions were fit by a multiple-Gaussian function. Mean values of
the first peaks are shown. EPSCs in (a) were evoked by high osmolarity.
EPSCs in (b) were electrically evoked in Sr2+. (c) Distribution of dendritically recorded EPSPs for a distal input location. Inset, distribution of
EPSPs simultaneously recorded at the soma of the same cell as in (c).
Note large number of apparent failures (arrow). Distribution was fit
(constrained to 0–0.6 mV) by a single Gaussian function. (d) Amplitude
of the smallest dendritically recorded events (EPSPs, or EPSCs, )
plotted as a function of synapse location. Amplitude of the smallest
somatically recorded events (EPSPs and voltage transients) plotted as a
function of synapse location (open circles). (e) Coefficient of variation
() and the σ2/x () as a function of location.
EPSC amplitude (mV)
Synaptic location
e
Synaptic location
ly regulated by common neuromodulators (such as acetylcholine, norepinephrine and serotonin)8,9,35. The release of these
neuromodulators at a localized region of the dendrite could
lower threshold, change temporal summation and produce a
very nonlinear form of integration36,37, perhaps even resulting in
the local initiation of dendritic spikes7,9,38. Such a regional, nonlinear type of integration would be transposed on top of the
otherwise global, spatially normalized input discussed above.
Therefore by removing the location dependence of synaptic
input, the neuron has extended the range of synaptic integration possible in the same dendritic arborization from a very
global non-varying type of processing to one that is perhaps
regional and highly nonlinear.
METHODS
Hippocampal slices (400 µm) were prepared from 6–12 week-old
Sprague–Dawley rats using standard procedures as described8. Individual neurons were visualized with a Zeiss Axioskop fit with differential
interference contrast (DIC) optics using infrared illumination. All neurons had resting membrane potentials between –63 and –75 mV. Wholecell patch-clamp recordings were made using two Dagan BVC-700 or a
combination of one Dagan and an Axopatch 200B amplifier in active
‘bridge’ mode. All synaptic currents were recorded with an Axopatch
200B. Data were acquired at 50 kHz and filtered at 3 or 5 kHz. The normal external recording solution contained 124 mM NaCl, 2.5 mM KCl,
1.2 mM NaH2PO4, 25 mM NaHCO3, 2.0 mM CaCl2, 1.5 mM MgCl2 and
10 mM dextrose, bubbled with 95% O2 and 5% CO2 at ∼35° C (pH 7.4).
In most cases, the external recording solution used during high osmolarity stimulation contained 0.5 mM CaCl2, 7.0 mM MgCl2 and 0.5–1 µM
tetrodotoxin (TTX). Whole-cell recording pipettes (somatic, 2 to 4 MΩ;
dendritic, 3.5 to 7 MΩ), were pulled from borosilicate glass. The internal
pipette solution consisted of 120 mM KMeSO4, 20 mM KCl, 10 mM
901
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articles
HEPES, 0.05 mM EGTA, 4.0 mM Mg2ATP, 0.3 mM Tris2GTP, 14 mM
phosphocreatine and 4 mM NaCl (pH 7.25 with KOH). A cesium-based
internal solution (120 mM Cs-gluconate replaced KMeSO4, and 1–2 mM
QX314 was added) was used for the generation of current–voltage plots.
Series resistance for somatic recordings was 6 to 20 MΩ, whereas that
for dendritic recordings was 15 to 40 MΩ for voltage recording or 12 to
25 MΩ for current recordings. Dendritic pipettes were coated with sylgard. Voltages have not been corrected for the theoretical liquid junction
potential (∼7 mV). Final concentrations of bicuculine methiodide (10
µM), APV (50 µM), TTX (0.5–1 µM, RBI) and A1 antagonist 8cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 µM) were made daily from
stock solutions dissolved in water. Error bars represent s.e.m., and the
number of cells (n) is given. ANOVA with Fishers post-hoc test was used
for statistical comparison.
The stimulation techniques used in this study were intended to
stimulate release from single terminals that were localized to an
approximate area of the dendrite (∼25 µm diameter). Bicuculine
methiodide (10 µM) and APV (50 µM) were present in the external
solutions during all recordings. When minimal stimulation techniques
were used21,22, a stimulating electrode was placed within 5–10 µm of
the dendrite under study (whole-cell pipettes coated with sylgard or
tungsten bipolar electrodes; A-M Systems, Carlsborg, Washington).
The close proximity of the stimulating electrodes made it very difficult, however, to find a stimulating spot where a wide range of stimulus currents could be used without a resulting increase in amplitude.
We used a template (composed of the rising and decaying exponential
equation shown in Methods) to isolate EPSCs from the recorded
region of the dendrite, and all selected EPSCs had similar synaptic
delay, rise and decay time. High osmolar external solution was pressure applied using a computer controlled pneumatic pump (Medical
Systems, Greenvale, New York) to a spot on the dendrite. (The pressure wave had a diameter of < 20 µm.)39,40. High osmolar external
solution consisted of normal external solution with the addition of
300 mM sucrose and 0.5–1 µM TTX and HEPES replacing NaHCO3.
Based on previous studies, we estimate that the final concentration
of external solution reached approximately 450 mOsM after mixing40.
A combination of these two techniques was used to stimulate in Sr2+containing solutions24. First, the external bath solution was changed
to one with 0.5 mM CaCl2 and 7 mM MgCl2, and a solution containing 0 mM added CaCl2, 10 mM SrCl2 and HEPES replacing NaHCO3
was pressure applied as above. The application pipette was used to
electrically stimulate at low stimulus intensities. In a few cases, Sr2+containing external solution was bath applied (NaHCO3 as buffer).
For analysis of EPSP/Cs, records were first smoothed (5 points averaged with a type of moving average algorithm, Igor, Wavemetrics) and
a threshold-crossing protocol (2 pA or 0.1 mV; ∼2× σnoise) was used to
automatically select events. The non-smoothed versions of these events
were then individually inspected and fit by the function ƒ(t) = a(1 –
exp(–t/τr))5exp(–t/τd), where a is a constant and τr and τd are the rise
and decay time constants. All events with a rise time slower than 0.8 and
0.4 ms for EPSP/Cs, respectively, were discarded as being too slow to have
occurred near the recording site (Fig. 3a). The spontaneous release rate
is very low at physiological temperatures in slices, so the number of these
contaminating events was very small (< 5%). To minimize the impact of
even these very infrequent spontaneous events, we triggered the averaging of somatic EPSPs only after the dendritic EPSP crossed the voltage
threshold during the dual recordings. Events were binned at 1 pA or 0.1
mV for amplitude distributions. Although there seemed to be some evidence of evenly spaced peaks in some distributions, insufficient numbers of events were collected (usually < 100) to allow an appropriate
determination24. The mean event rate (determined from interval distributions) was less than 10 Hz for high osmolar solution application and
less than 5 Hz for events evoked in Sr2+. Using these rates, the probability of two events occurring within 1 ms of each other is ∼5 × 10–6 for high
osmolar solution, and ∼2.5 × 10–6 for stimulation in Sr2+ according to
Poisson statistics41. In most figures, the data were arbitrarily fit by either
an exponential or linear function. If they could not be fit by one of these
functions, then they were arbitrarily fit by a polynomial function.
The neurophysiological modeling program NEURON was used to
simulate a reconstructed CA1 cell (provided by B.J. Claiborne and
902
D.B. Jaffe). All synaptic input was modeled as an AMPA receptor-like
conductance change using an alpha function (time to peak, 1 ms) and a
reversal of 0 mV. The passive parameters Rm and Ra were varied as indicated, with Cm always set to 1 uF/cm2.
ACKNOWLEDGEMENTS
We thank M. Carruth for technical assistance, M. Vollrath for comments on the
manuscript and D. Johnston for discussions throughout the study. This work was
supported by National Institute of Health grants NS35865 and NS39458 and by
the Alfred P. Sloan Foundation.
RECEIVED 17 MAY; ACCEPTED 25 JULY 2000
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