1Veterans Affairs Puget Sound Health Care System, Seattle 98108 and 2Department of Neurology and 3Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195
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ABSTRACT |
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Nettleton, Jilda S. and William J. Spain. Linear to Supralinear Summation of AMPA-Mediated EPSPs in Neocortical Pyramidal Neurons. J. Neurophysiol. 83: 3310-3322, 2000. It has been hypothesized that voltage-sensitive conductances present on the dendrites of neurons can influence summation of excitatory postsynaptic potentials (EPSPs) and hence affect how neurons compile information. Greater than linear summation of EPSPs has been postulated to facilitate coincidence detection by cortical neurons. This study examined whether the summation of subthreshold AMPA-mediated EPSPs generated on layer V neocortical pyramidal neurons in vitro was linear and if any nonlinearities could be attributed to dendritic conductances. Evoked EPSPs (1-12 mV) were recorded somatically by means of intracellular sharp electrodes in the presence of 100 µM amino-5-phosphonopentanoic acid (AP-5) and 3 µM bicuculline. Two independent EPSPs were evoked by a stimulating electrode in layer I and another in layers III-V. The areas of stimulation were isolated from each other by a horizontal cut below layer I. By subtracting the algebraic sum of the individual EPSPs from the evoked response when both EPSPs were evoked simultaneously, we determined that they summed linearly to supralinearly. Supralinear summation was more likely when the soma was hyperpolarized by DC current injection. Summation was predominantly linear when postsynaptic conductances (i.e., Na+ and Ca2+) were blocked with intracellular QX-314. The supralinear summation of EPSPs (without QX-314) decreased as the time between inputs was increased from 0 to 30 ms. To determine the role of dendrites in nonlinear summation, we substituted a current pulse (simulated EPSP) delivered at the soma for either or both of the evoked EPSPs. Simulated EPSPs combined with either an evoked EPSP or another simulated EPSP showed significantly less supralinear summation than two evoked EPSPs, indicating that the dendritic conductances were largely responsible for the observed supralinear summation.
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INTRODUCTION |
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Neurons in cerebral cortex receive a continuous
barrage of both excitatory and inhibitory synaptic inputs. There are
estimated to be 3,000-10,000 synaptic inputs on a cortical neuron
(Peters 1994) of which a considerable fraction may be
active at any one time. How does the neuron integrate these individual
events into an output firing pattern? According to the Rall model in
which the dendrites are assumed to be passive, EPSPs would sum either linearly or sublinearly when there is reduction in the driving force
for synaptic conductances (Rall et al. 1967
). Dendrites are not passive but instead possess voltage-sensitive conductances that
could affect synaptic integration (for review, see Yuste and
Tank 1996
). Dendrites have been shown to generate regenerative Na+- and Ca2+-dependent
potentials (Amitai et al. 1993
; Kim and Connors
1993
; Llinas and Sugimori 1980
;
Pockberger 1991
; Reuveni et al. 1993
; Schiller et al. 1997
; Spruston at al.
1995
; Stuart and Sakmann 1994
; Stuart et
al. 1997
; Wong et al. 1979
). Additionally,
dendritic conductances have been shown to modify synaptic currents
(Lee and Heckman 1996
; Schwindt and Crill 1995
,
1997
). Pyramidal neurons may have a nonhomogeneous distribution
of ionic conductances that could potentially affect how EPSPs at
different locations are integrated. Studies in both neocortical and
hippocampal pyramidal neurons indicate that there is an uneven
distribution of Ca2+ channel types along the
apical dendrite (Hell et al. 1993
; Johnston et
al. 1996
; Reuveni et al. 1993
; Schiller
et al. 1995
; Westenbroek et al. 1992
;
Yuste et al. 1994
). There is also evidence that neurons may express uneven distributions of other ion channel types as well
(Maletic-Savatic et al. 1995
; Sheng et al.
1994
; Stuart and Spruston 1998
;
Westenbroek et al. 1989
).
How could these conductances affect integration of subthreshold
excitatory postsynaptic potentials (EPSPs)? Evoked subthreshold synaptic potentials can activate Na+ and
Ca2+ channels (Magee and Johnston
1995a,b
) and cause calcium influx into dendrites (Magee
et al. 1995
; Markram and Sakmann 1994
;
Schiller et al. 1997
). Na+ and
Ca2+ conductances could cause supralinear
summation by increasing the size of EPSPs (Gillessen and
Alzheimer 1997
; Lipowsky et al. 1996
). In
contrast, dendritically located K+ channels
decrease the size of the EPSP in hippocampal pyramidal neurons
(Hoffman et al. 1997
) and decrease synaptic current in neocortical neurons (Schwindt and Crill 1997
). In
cultured hippocampal pyramidal neurons,
IA causes sublinear summation of
synaptic inputs (Cash and Yuste 1998
). How EPSPs sum may
depend on the relative quantity of Na+,
Ca2+, and K+ conductances
activated by EPSPs.
Ionic conductances are also time dependent, the relative timing of
inputs could have an effect on how much they are able to affect each
other. Some modeling studies indicate that layer V neurons could act as
coincidence detectors and that only inputs within a millisecond of each
other are able to interact with each other to bring the neuron to
firing threshold (Konig et al. 1996; Softky and
Koch 1993
). Other studies propose that neurons operate in an
integrate and fire mode (Shadlen and Newsome 1994
). The time dependence of excitatory synaptic summation in pyramidal neurons
has yet to be measured experimentally.
The goal of this study was to determine if layer V pyramidal neurons
sum their synaptic inputs in a linear or nonlinear fashion. We found
that subthreshold EPSPs can sum supralinearly in layer V pyramidal
neurons due to postsynaptic, QX-314-sensitive conductances, and we
determined that dendritic conductances contributed to the supralinear
summation. These results give new information about how neurons
integrate information. Some of the results have been presented
previously in abstract form (Nettleton and Spain 1996).
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METHODS |
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Tissue preparation
Slices of sensorimotor cortex were obtained from 24- to
90-day-old Sprague-Dawley rats of either sex as described previously (Cerne and Spain 1997). Briefly, rats were anesthetized
with intraperitoneal injections of ketamine and xylazine. Once the
animal was areflexive to strong foot pinch, the carotid arteries were
cut and sensorimotor cortex was excised. Either 350- or 400-µM
coronal slices were cut on a Vibratome and maintained at 33°C in a
carbogenated (95% O2 and 5%
CO2) Ringer solution containing (in mM) 130 NaCl,
3 KCl, 2 CaCl2, 2 MgCl2,
1.2 NaH2PO4, 26 NaHCO3, and 10 dextrose (pH, 7.4). For recording,
slices were transferred to a submerged-type chamber (volume = 0.5 ml) and perfused at 2.5 ml per minute with the carbogenated Ringer
solution (34 ± 1°C).
Intracellular recording
Recordings were performed intracellularly on the presumed somata
of layer V pyramidal neurons. Recording electrodes were made from
borosilicate glass (OD/ID = 1.0 mm/0.58 mm, Sutter Instruments, Novato, CA) pulled on a Flaming/Brown micropipette puller P-87 (Sutter
Instruments) and filled with 2.7 M KCl and 1% biocytin. For some
experiments, electrodes also contained 50 mM QX-314 (Alomone Labs or
Sigma). Resistance was 25-60 M for all electrodes.
Recording was done in current-clamp using the bridge mode of an AxoClamp amplifier (Axon Instruments, Foster City, CA). Current and membrane potential were low-pass filtered (5 kHz) and recorded on videocassettes using pulse-code modulation (44 kHz) (Neuro-Corder DR-890, Neurodata, New York). All stimulating protocols, data collection, and analyses were computerized using Igor Pro (Wavemetrics, Lake Oswego, Oregon) on a Quadra 800 (Apple Computers, Cupertino, CA) and an ITC16 computer interface (Instrutech, Great Neck, NY) using customized programs. During the experiment, both evoked and simulated EPSPs were digitized at 0.1 ms. Measured values are reported as the means ± SE. Unless otherwise stated, statistical comparisons were performed using a two-tailed Student's t-test with the significance criterion set at P < 0.05. Gaussian curves were fit to histograms using a nonlinear, least-squares fitting routine (Levenberg-Marquardt algorithm).
Evoked and simulated EPSPs
Figure 1 shows the method used to
isolate two sites of stimulation so that stimulation at one site did
not affect fibers stimulated by the other site. A small piece of razor
blade was guided by micromanipulator to make a cut in the slice just
below layer I. The cut extended from near the apical dendrite of the
recorded neuron to the lateral edge of the slice (Fig. 1A).
The distal (Ds) stimulating electrode was placed in layers I-II (above
the cut), and the more proximal (Px) stimulating electrode was placed below the cut in lower layer III to upper layer V. The Ds stimulating electrode was placed 200-500 µm from the middle of the distal dendritic tuft. The Px stimulus was 200-500 µm from the main trunk of the apical dendrite and 150-300 µm toward the pia from the recording electrode. Both stimulating electrodes were 150-350 µm
lateral from the end of the cut nearest to the apical dendrite. These
two sites were chosen because they were far enough apart to prevent one
stimulus from affecting the other, yet both would still stimulate
inputs to the apical dendrite (Cauller and Connors 1994;
Ichinose and Murakoshi 1996
; White
1989
). In most experiments, fine-point monopolar
stainless steel electrodes were used. In a few experiments, concentric
bipolar electrodes were used (FHC, Bowdoinham, ME). Each stimulating
electrode was separately driven by a constant-current stimulus isolator
(Model A365, WPI, Sarasota, FL) using either a 1- or 0.2-ms TTL
pulse at a frequency of 0.33 Hz. The current amplitude (range, 1.7-70
µA) was adjusted so that the amplitude of the isolated EPSPs were
1
mV but below the amplitude that evoked large regenerative
depolarizations when both stimuli were given together. These
regenerative events typically had a delayed latency of
30-40 ms and
were a consequence of having inhibition partially blocked
(Chagnac-Amital and Connors 1989
) since they did not
occur prior to the addition of bicuculline to the bath.
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We looked for possible interactions between the two stimulus sites by
comparing responses from paired Px and Ds stimuli with responses from
paired pulses at one of the stimulus sites. Paired-pulse facilitation
and paired-pulse depression result from stimulating the same fibers
twice in 50-70 ms (Fisher et al. 1997; Zucker 1989
). If the two stimuli in these experiments are stimulating some of the same fibers, then paired-pulse depression or paired-pulse facilitation should be observed when the Px and Ds stimuli are 50-70
ms apart. While in some cases we found paired pulse facilitation for
either Px-Px or Ds-Ds paired stimuli, we did not observe any paired
pulse facilitation or paired pulse depression when the Px and Ds
stimuli were delivered 50-70 ms apart.
Experimental protocols for stimulation were as follows: first the Ds and Px stimuli were given individually 10-15 times each at a rate of 0.33 Hz. The two stimuli were then given together 15-20 times (with the interstimulus delay ranging from 0 to 30 ms). Finally the individual Px and Ds stimuli were again delivered 10-15 times each. Analysis was performed on the averaged 10-20 responses to individual Px, Ds, and simultaneous Px plus Ds evoked EPSPs. For each set of stimulus conditions, we checked for nonstationarity of EPSP size by comparing the integral of the algebraic sum of the EPSPs evoked at the start of each set to the same integral from EPSPs evoked at the end of each set. Data were only used from EPSPs whose summed integral changed by <15%.
To isolate AMPA-mediated EPSPs, slices were perfused continuously with
3 µM bicuculline (to block GABAA-mediated
responses) and 100 µM amino-5-phosphonopentanoic acid (AP-5) [to
block N-methyl-D-aspartate (NMDA)-mediated
responses]. The slices were perfused for 20 min before summation
experiments were performed. These neurons had little or no
GABAB-mediated responses at the low stimulus
strengths used in this study (Benardo 1994
; van
Brederode and Spain 1995
). Furthermore, the EPSPs elicited in
these experiments were completely blocked by 20-60 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX; Tocris Cookson, St. Louis, MO).
Residual EPSPs or inhibitory postsynaptic potentials (IPSPs) were not
seen at holding potentials of
69 to
97 mV.
In some experiments, simulated EPSPs were created by intracellular
current injection, I, as a function of time, t,
according to the equation (from Otis et al. 1993)
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Histology
After recording, the tissue was fixed using 4% paraformaldehyde
in a phosphate buffer solution containing (in mM): 19 NaH2PO4, 83 Na2HPO4, 150 NaCl, and 3 KCl. Once fixed, tissue was either sliced further or left in
whole-mount sections. The tissue was processed using a diaminobenzidene
reaction to biocytin (Horikawa and Armstrong 1988) or
using a fluorescent probe that cross reacts with biocytin. For the
fluorescent imaging, neurons were visualized after immersion in 1:100
dilution of Texas Red, Texas RedX or Oregon Green (Molecular Probes,
Eugene, OR) for 4-7 days.
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RESULTS |
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The results are based on intracellular recordings from 56 neurons
that were recovered histologically and shown to be layer V pyramidal
neurons with an intact apical dendrite reaching into layer 1 past the
cut (Fig. 1A). For 34 neurons recorded using electrodes
containing 2.7 M KCl and biocytin, the mean resting membrane potential
was 74 ± 1 mV and mean input resistance was 21 ± 1 M
(measured at the end of 5- to 10-mV hyperpolarizing responses evoked by
1-s negative current pulses). All neurons fired action potentials
that overshot 0 mV. The 34 neurons were classified as regular spiking
neurons (20) or bursting neurons (14, neurons that fired
3 action
potentials on an initial depolarizing hump) (Connors and Gutnick
1990
). No relationship between neuron type and EPSP summation
was observed.
Linear to supralinear summation of EPSPs
As shown in Fig. 1, two stimulating sites, one distal (Ds) and the
other proximal (Px), were used to evoke EPSPs in layer V pyramidal
neurons. To determine if the Ds- and Px-evoked EPSPs summed linearly,
the method shown in Fig. 2 was used. The
integral of the EPSP generated in response to the simultaneous
stimulation at the Px and Ds sites was divided by the integral of the
algebraic sum of the individual Px- and Ds-evoked EPSPs to give a
summation ratio (SR). In a given neuron, the latency of the Ds-evoked
EPSP (4.9 ± 0.2 ms) was always longer than the latency of the
Px-evoked EPSP (2.9 ± 0.1 ms), similar to previous observations
(Cauller and Connors 1994) (latencies were measured from
the stimulus onset to the initial rise of the EPSP). Therefore the
integrals began 1 ms after the start of the distal EPSP and were
limited to a 15-ms interval in order not to include late polysynaptic
events. During late polysynaptic activity, the Ds and Px stimulus might not be isolated from each other because of the possibility of both
stimuli acting on common neurons presynaptic to the recorded neuron
through polysynaptic connections.
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Figure 3C shows the distribution of SR values measured in layer V pyramidal neurons. The mean SR of 1.12 ± 0.02 (n = 74 SR measurements from 23 cells) was significantly >1, the expected SR value of linear summation. There was also a wide range of SR values (0.81-1.73), and the distribution was skewed in the direction of supralinear summation. In some cases in which the SR was greater than linear, there was an inflection in the rising phase of the summed EPSP (arrow in Fig. 3A) suggesting that supralinearity may be due to activation of a regenerative conductance. In these cases, the peak of the summed EPSP was delayed with respect to the peak of the algebraic sum of the Px- and Ds-evoked responses. In other cases where there was supralinear summation, the summed EPSP was a scaled-up version of the algebraic sum (e.g., Figs. 2, 4A, and 8A). A plot of the delay of the peak of the summed EPSP versus SR (Fig. 3C, inset) shows an increase in SR as a function of delay indicating a direct relation between a regenerative process and the amount of supralinear summation.
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Supralinear summation is postsynaptically determined
A goal was to determine if postsynaptic conductances contributed
to nonlinear summation of synaptic inputs. Therefore the SR was also
determined for a set of neurons filled with QX-314. QX-314, a
permanently charged lidocaine derivative, has been shown to internally
block several voltage-sensitive conductances: Na+
(Strichartz 1973), some types of
K+ (Andreasen and Hablitz 1993
),
Ca2+ (Talbot and Sayer 1996
), and
Ih (Perkins and Wong
1995
). To allow QX-314 to diffuse into the dendrites, data were
gathered after neurons had been penetrated for >40 min (after which
the I-V relationship showed no further changes). In the
presence of QX-314, neurons ceased firing action potentials, input
resistance increased, and the steady-state I-V relation was
linearized around resting potential. The mean input resistance for the
23 neurons recorded with QX-314 electrodes was 67 ± 11 M
. This
was significantly larger than the value obtained from neurons recorded
without QX-314 in the electrode (control neurons). The mean resting
potential (
69 ± 1 mV) was slightly but significantly more
depolarized in QX-314-filled neurons than in control neurons. The range
of stimulation intensities used to evoke EPSPs in QX-314 filled neurons
was 2.7-7.7 µA for the Px stimulus and 3.6-40 µA for the Ds
stimulus. The stimulus amplitudes did not differ significantly from
those used in control neurons (Px = 1.7-9.5 µA; Ds = 4-60
µA). The increase in input resistance (with QX-314) would be expected
to cause larger EPSPs. However, the range of EPSP sizes evoked in
QX-314 versus control neurons were not significantly different.
Presumably, the change in input resistance is offset by the block of
conductances which amplify the EPSPs (e.g., Deisz et al.
1991
). By blocking many of the voltage-sensitive conductances
thought to be responsible for nonlinear summation, this method allowed
us to determine how these conductances affect EPSP summation. One
limitation of these experiments is that they may not account for all
nonlinear summation, particularly sublinear summation because QX-314
does not necessarily block all voltage-sensitive conductances
completely nor would it block any sublinear summation caused by changes
in driving force or synaptic conductance changes.
The distribution of SR values obtained with QX-314 in the recording electrode is shown in Fig. 3D. On average the EPSPs summed linearly with a mean SR of 1.00 ± 0.02 (n = 34, 14 neurons), which was significantly smaller than the SR measured in control neurons. Comparing the distribution of SR values measured in neurons with and without QX-314 revealed other differences. First, the range of SR values measured with QX-314 electrodes was smaller (0.82-1.33). Second, the distribution of SR values from QX-314 filled neurons was well fit by a Gaussian curve with a peak at SR = 0.99 similar to the mean (Fig. 3D). In comparison, the distribution of SR values from control neurons was skewed to the right. Based on the Gaussian fit to the QX-314 data, an upper 95% confidence limit of 1.16 was determined. From the SR values in neurons without QX-314, 23% were above this 95% cutoff limit and were thus considered to show QX-314 sensitive supralinear summation. Figure 3A shows an example of EPSPs with supralinear summation recorded without QX-314, while 3B shows typical EPSP summation from a QX-314-filled neuron. The smaller mean and more restricted range of SR values found in QX-314-filled neurons indicates that QX-314 sensitive conductances are contributing to supralinear summation.
Neurons recorded without QX-314 could not be grouped according to their ability to perform linear or supralinear summation. Instead, a wide range of SR values were obtained for different experimental conditions (i.e., changes in holding potential and/or stimulus strength) within a given neuron. We observed that for neurons with at least three SR measurements, the variation of SR was at least half the variance in SR for all neurons. Additionally, although only 23% of all SR values were >1.16, 35% of all neurons had at least one SR value >1.16. Therefore the most supralinear SR values did not belong to a subset of neurons. Since there did not appear to be a strong neuron-specific effect on SR values, all measurements were pooled for further analysis with some neurons contributing more than one SR value.
Summation ratio dependence on membrane potential and EPSP size
Because the summation of EPSPs is affected by QX-314-sensitive
conductances, conditions that change the state of those active conductances (e.g., membrane potential) would be expected to change the
amount of summation observed. We therefore measured the relationship between membrane potential and SR. SR increased (e.g., Fig.
4A) in 8 of 10 neurons in
which the stimuli were held constant and the holding potential was
hyperpolarized from between 71 and
81 mV to between
82 and
97
mV (mean increase = 0.10 ± 0.03 SR units per 10 mV
hyperpolarization, range = 0.26 to 0.03 SR units per 10 mV). In
one neuron, SR did not change, and in another, SR decreased 0.04 SR
units per 10 mV of hyperpolarization. The entire population of SR
values is shown graphically in Fig. 4B where SR values are
plotted as a function of the membrane holding potential. The data are
fit to a linear regression with a slope of
0.09 ± 0.02 SR units
per 10 mV, similar to what was found for individual neurons. The slope
of the regression is significantly different from zero with an
R value of 0.48, indicating that 23% of the variation in SR
values can be accounted for by changes in holding potential. Of EPSPs
with a holding potential more negative than
84 mV, 42% had SR
>1.16, while for more depolarized EPSPs only 10% were in this
category.
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In contrast, SR values from neurons recorded with conductances blocked
by QX-314 did not vary significantly with holding potential in
individual neurons (n = 5). Likewise, for all EPSPs
recorded with QX-314 (Fig. 4B), linear regression of SR
versus holding potential was not significantly different from zero. The
slope of 0.005 ± 0.017 SR units per 10 mV measured for the
QX-314-filled neurons was significantly different from the slope
measured in control neurons. Therefore we conclude that the increase of
SR with increasing hyperpolarization is not simply due to a change in
the driving force for AMPA-mediated conductance but results from
QX-314-sensitive voltage-dependent conductance.
The amplitude of EPSPs would also be expected to affect the amount of
nonlinear summation since larger EPSPs will activate voltage-dependent
conductances to a different degree than smaller EPSPs. Unlike holding
potential, EPSP amplitude had no clear effect on SR over the range of
amplitudes shown in Fig. 5. In individual neurons where the EPSP amplitude was systematically changed, the dependence of SR on EPSP size was variable. In the five cases where the
Px stimulus strength was increased systematically while holding
potential and Ds stimulus strength were held constant, four showed an
increase in SR while one decreased (average: 0.31 ± 0.25 SR units per 10 mV; range: 0.59 to 0.93 SR units per 10 mV). In
four cases where Ds stimulus strength was systematically increased, the
SR was not significantly changed (average: 0.04 ± 0.07 SR units per 10 mV; range:
0.19 to 0.16 SR units per 10 mV,
n = 4).
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The relationship between SR and a larger range of EPSP amplitudes is
shown in Fig. 5 where all SR values are pooled and plotted as a
function of Px amplitude, Ds amplitude, and Px plus Ds amplitudes. For
all three plots, linear regression reveals slopes of 0.16 ± 0.009, 0.08 ± 0.008, and 0.01 ± 0.005 SR units per 10 mV,
respectively. None of these slopes are significantly different from
zero. Since the effect of holding potential could have masked an effect
of EPSP size on SR, we also used multiple regression to examine any possible dependence of SR on holding potential and/or EPSP amplitude. Multiple regression analysis revealed that only holding potential had a
significant effect on SR in neurons recorded without QX-314.
One limitation of trying to determine the effect of EPSP size on SR was
that the EPSP amplitude could only be varied over a narrow range in any
one neuron. This was due to inhibition being largely blocked, so small
increases in stimulus strength resulted in large polysynaptic
potentials (Chagnac-Amitai and Connors 1989) when the
two stimuli were given together. Another possible explanation for the
surprising lack of dependence of SR on EPSP amplitude is that, while
larger EPSPs may activate more Na+ and
Ca2+ conductance (which would be expected to lead
to an increase in SR), larger EPSPs might increase the relative
contribution of voltage-sensitive K+ conductances
to synaptic integration. Potassium conductance alone would decrease SR
(Cash and Yuste 1998
; Margulis and Tang
1998
; Urban and Barrionuevo 1998
) and counter
further increases in SR as a function of depolarization activated
inward currents.
Effect of time between EPSPs on summation ratio
Activation of voltage-dependent conductances by EPSPs could either
improve coincidence detection (Bernander et al. 1991;
Konig et al. 1996
; Softky and Koch 1993
)
or favor temporal summation, depending on the characteristics of the
conductance. For example, a conductance with rapid activation and
inactivation kinetics could cause supralinear boosting mainly to EPSPs
that are nearly coincident. In contrast, a noninactivating inward
conductance with slow deactivation kinetics could favor supralinear
summation of EPSPs separated in time and thus enhance temporal
summation. Therefore to gain insight into how nonlinear summation might
influence information processing, we measured SR as a function of the
time between the Ds and Px stimuli.
Examples of experiments to determine the effect of a delay between
synaptic inputs on SR are shown in Fig.
6A. For the neuron recorded
without QX-314, the SR decreased from 1.13 to 1.05 as the time
(t) between the Ds and Px stimuli increased from 5 to 30 ms. On the other hand, in a QX-314-filled neuron, the SR was close to
linear (1.0) for all
t. In nine individual control
neurons in which EPSP size and holding potential were held constant
while
t was increased, six had the largest SR at 0 or 5 ms, one had its peak SR at 10 ms, and two neurons had SR values that
were independent of changes in
t. Figure 6,
B-D, shows the SR distribution histograms for all control
and QX-314-filled neurons in which the two inputs were separated by 5, 10, and 30 ms. A majority of summed EPSPs (80%) had the Px EPSP
preceding the Ds EPSP. In the remaining, the Ds stimulus was given
first. Both groups were analyzed together since no correlation between
stimulus order and EPSP summation was observed. Similar to what was
observed for
t = 0 (e.g., Fig. 3), the distribution
of SR's in control neurons were skewed to the right for
t's equal to 5 and 10 ms. In the presence of QX-314 and
at the same
t, the distribution of SR values was relatively normal.
For
t of 30 ms, the distribution of SR values was close
to normal for both groups of neurons. At all
t's and for
both groups of neurons, measurements were taken over the same range of
holding potentials and EPSP sizes. As the time between inputs
decreased, the effect of QX-314 on SR values increased, indicating that
an active conductance caused the SR to increase in a time-dependent
fashion.
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Since holding potential was shown to affect SR summation at
t = 0, we examined the relationship between SR and
t for EPSPs with holding potentials positive to
85 mV (depolarized
EPSPs) separately from EPSPs with holding potentials equal to or more negative than
85 mV (hyperpolarized EPSPs). Figure
7, A and B, shows
plots of the relation of SR to
t for all depolarized and hyperpolarized EPSPs, respectively. At hyperpolarized potentials, SR
decreased more dramatically with time between inputs than at potentials
closer to rest. Such results imply that there is a time-dependent
component to supralinear summation that is more pronounced at
relatively hyperpolarized holding potentials (i.e., less than or equal
to
85 mV).
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While a QX-314-blockable conductance contributes to a SR >1, there are
other possible factors affecting the relationship between SR and time.
Factors such as synaptic conductance change, presynaptic interactions
between the stimuli, or postsynaptic conductances not blocked or only
partially blocked by QX-314 might affect how EPSPs sum in QX-314-filled
neurons. Consistent with this idea, we observed that in the presence of
QX-314, not all EPSPs summed linearly at all t's. In
particular, the mean SR peaked at
t = 5 ms for both
the hyperpolarized EPSPs and the EPSPs closer to resting membrane
potential. Such behavior is consistent with a non-QX-314-blockable
component contributing to supralinear summation when the inputs are
within 5 ms of each other combined with a reduction in driving force
and/or a synaptic conductance change decreasing EPSP summation when the
inputs are simultaneous.
To examine only the contribution of the QX-314-blockable conductances
to the SR versus t relationship, we plotted the
difference in mean SR (
SR) between neurons recorded with and without
QX-314 as a function of
t (Fig. 7C). At both
hyperpolarized and resting membrane potentials, there was a clear
decrease in
SR with
t. At membrane potentials more
positive than
85 mV, SR decreased linearly (slope =
0.002 ± 0.001 SR units/ms) as the time between inputs increased, until at
t = 30 ms,
SR was not significantly different
from zero. For the hyperpolarized EPSPs, the decrease in
SR with
time between stimuli was well fit by a single exponential with a time
constant of 15 ± 8 ms. These results indicate that there is a
QX-314-sensitive, time-dependent component to supralinear summation
that is altered by changes in membrane potential.
Dendritic conductances cause supralinear summation of EPSPs
Previous studies indicate that voltage-sensitive conductances on
the dendrites play an important role in synaptic integration (for
reviews, see Johnston et al. 1996; Yuste and Tank
1996
). The hypothesis that dendritic conductances contributed
to the supralinear summation that we observed in Fig. 3 was tested by generating simulated EPSPs with current pulses injected through the
recording electrode at the soma (see METHODS). The
simulated EPSPs were then substituted for one or both of the evoked
EPSPs in the experimental protocol shown in Figs. 1 and 2. For all SR values measured from simulated EPSPs, there was no delay between the
two stimuli. Figure 8A shows
an example of a neuron in which the simulated EPSPs closely
approximated the time course and amplitude of the evoked EPSPs. The
evoked EPSPs summed supralinearly with an SR of 1.18. When simulated
EPSPs were substituted for either the Px, Ds, or both evoked EPSPs, SR
values were close to linear (0.99, 1.05, and 1.02, respectively). For
12 neurons in which simulated EPSPs were used, the amount of summation
did not depend on whether the simulated EPSPs were substituted for
either the Px or Ds or both of the evoked EPSPs. There was no
significant difference among the mean SR measured for each of the three
groups (simulated Px with evoked Ds: 1.04 ± 0.01, n = 30; evoked Px with simulated Ds: 1.03 ± 0.01, n = 13; both Px and Ds simulated: 1.06 ± 0.02, n = 22). In the same group of neurons, two evoked EPSPs were found to have significantly larger SR: 1.16 ± 0.04, n = 30 (only SR values from EPSPs that were at the same
holding potentials as the simulated EPSPs were included in this mean;
1-way ANOVA, P < 0.05 for all group comparisons).
Figure 8B compares the SRs from two evoked EPSPs to SRs from
well-matched simulated EPSP. In the five cases where the two evoked
EPSPs yielded SR values above the QX-314 limit of 1.16, their matched
simulated EPSPs yielded SR values close to linear. For the remaining SR
measurements, evoked EPSPs yielded larger or equivalent SR values
compared with those SR values measured using simulated EPSPs. This
demonstrates that although simulated EPSPs created at the soma have SR
values >1, the simulated EPSPs are not sufficient to create the same amount of supralinear summation as those obtained from two evoked EPSPs.
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Conductances near or in the soma may also contribute to supralinear summation. To assess this effect, we compared SRs from summation with simulated EPSPs obtained from neurons recorded without to those recorded with QX-314 electrodes (examples are shown in Fig. 9, A and B, respectively). Figure 9, C and D, shows the distribution of all SR values from control and QX-314-filled neurons, respectively. As seen for evoked EPSPs (Fig. 3), the SR values obtained from control neurons are skewed to the right while the SR values obtained from QX-314-filled neurons have a normal distribution. Since there was no dependence of summation on which EPSP was simulated (or if both were simulated), all SR from simulated EPSPs were pooled. In QX-314-filled neurons, simulated EPSPs combined with an evoked or another simulated EPSP had an average SR value of 0.98 ± 0.01 (n = 20) while in neurons recorded without QX-314, the average SR was significantly larger (1.04 ± 0.01, n = 65; P < 0.02). Thus QX-314-sensitive conductances near or in the soma also affected the degree of linearity of EPSP summation.
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Some insight into the relative contribution of somatic versus dendritic conductances can be gained by comparing Fig. 3 with Fig. 9 (note the difference in the horizontal axis scale). In neurons without QX-314, the overall mean SR from two evoked EPSPs (1.12 ± 0.02 ± n = 74) was significantly larger than the mean SR from summation with one or more simulated EPSPs (1.04 ± 0.01, n = 65), demonstrating that dendritic conductances contribute to larger SR values. In contrast, QX-314-filled neurons had a mean SR for evoked EPSPs (0.99 ± 0.01) that was not significantly different from the mean SR for simulated EPSPs (0.98 ± 0.01). Thus QX-314 eliminated both the somatic and dendritic conductances responsible for supralinear summation. Such a difference indicates that a postsynaptic dendritic conductance is responsible for a majority of the supralinear summation observed in these experiments.
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DISCUSSION |
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This study provides evidence that voltage-sensitive conductances
in dendrites play a role in EPSP summation. Models of passive dendrites
predict that synaptic inputs will sum sublinearly if the signal from
one input affects the synaptic driving force of other inputs
(Rall 1977; Rall et al. 1967
) or if
synaptic conductance changes cause a reduction in input resistance
(Bernander et al. 1991
). Early support for the effects
of synaptic driving force came from measurements of sublinear EPSP
summation in motoneurons (Burke 1967
). There is now,
however, substantial evidence that many types of neurons contain
voltage-sensitive conductances throughout their dendritic trees (for
review, see Johnston et al. 1996
; Yuste and Tank
1996
). Currently there is insufficient characterization of
dendritic conductances to make an assumption free empirical model that
allows for accurate predictions of how the active properties of
pyramidal neuron dendrites will influence the spatial and temporal summation of EPSPs. Recent studies on hippocampal pyramidal neurons that used an approach similar to this study have found linear to
sublinear summation (Cash and Yuste, 1998
; Urban
and Barrionuevo 1998
). However, one study found a switch from
supralinear to sublinear summation, which depended on the timing of
paired pulses of glutamate release at one dendritic location
(Margulis and Tang 1998
). In contrast, we found that
summation of two spatially separated sets of AMPA-mediated synaptic
inputs onto layer V neocortical pyramidal neurons is linear to
supralinear and that supralinearity is caused by dendritically located
postsynaptic conductances. The amount of nonlinear summation is
affected by the time interval between inputs and the membrane potential
of the postsynaptic neuron.
Supralinear summation is due to postsynaptic mechanisms
When supralinear summation occurred it was primarily due to postsynaptic mechanisms. First, intracellular QX-314 reduced the mean SR to linearity. A presynaptic mechanism for supralinear summation would have been unaffected by intracellular QX-314. Additionally, in QX-314-filled neurons, summation using simulated EPSPs was similar to summation with evoked EPSPs. If presynaptic mechanisms were affecting summation, simulated EPSPs would have had a different mean SR than evoked EPSPs. Finally, paired pulses indicated that the two stimuli were stimulating separate sets of fibers (see METHODS).
Dendritic conductances contribute to supralinear summation
Our results show that the QX-314-sensitive conductances underlying
supralinear summation are activated by dendritic events. If the soma
region itself contributed to large amounts of supralinear summation, we
would expect the SR values measured with simulated EPSPs to be similar
to SR values measured with evoked EPSPs. Instead, simulated EPSPs had
less supralinear summation compared with evoked EPSPs. The Ds stimulus
likely stimulated the distal tuft of the apical dendrite while the Px
stimulus likely stimulated more proximal portions of the apical
dendrite and/or parts of the basal dendrites due to horizontal cortical
pathways (Cauller and Connors 1994; Ichinose and
Murakoshi 1996
; Thomson and Deuchars 1994
;
White 1989
). The amplitude of an evoked EPSP
attenuates as it spreads from the apical dendrite to the soma
(Stuart and Sakmann 1995
). Also the amplitude of a
simulated EPSPs injected at the soma attenuates by
80% at a point
600 µm distal on the apical dendrite (Stuart and Spruston
1998
). Therefore a somatically injected simulated EPSP would
not be expected to activate as much dendritic Na+
or Ca2+ conductance as an evoked EPSP that caused
the same amount of somatic depolarization as the simulated EPSP.
Another consideration is that in these experiments, which utilized
sharp electrodes, the input resistance of layer V pyramidal neurons was
22 ± 2 M, while whole cell recording from the same aged rats
with patch electrodes yielded an input resistance of 77 ± 5 M
(from Cerne and Spain 1997
) (temperature = 33 ± 1° C). The 3.5-fold difference in input resistance is indicative
of a somatic shunt resulting from the use of sharp electrodes
(Spruston and Johnston 1992
). Due to this shunt, somatic
conductances may contribute less to EPSP summation than they would
normally. Thus under the experimental conditions employed in this
study, only the supralinear summation in the dendrites was readily
apparent, while the somatic component was minimal.
What conductances underlie supralinear summation?
A QX-314-sensitive conductance underlies the cases of supralinear
summation. QX-314 blocks several conductances
(Na+, Ca2+,
Ih, and K+
conductances) (Andreasen and Hablitz 1993; Issac
and Wheal 1993
; Nuñez and Buño 1992
;
Perkins and Wong 1995
; Stafstorm et al. 1985
; Talbot and Sayer 1996
).
Ih and most K+
conductances would be expected to cause attenuation of EPSPs and
sublinear summation (Cash and Yuste 1998
; Hoffman
et al. 1997
; Magee 1998
; Margulis and
Tang 1998
; Schwindt and Crill 1997
;
Stuart and Spruston 1998
; Urban and Barrionuevo
1998
; but see Wessel et al. 1999
). We found
summation to be linear to supralinear with a direct relation between
the amount of supralinear summation and the appearance of a
regenerative component to the summed EPSPs (Fig. 3A and
inset in C). Furthermore hyperpolarization of
membrane potential increased supralinear summation. Membrane
hyperpolarization will remove inactivation of both T-type
Ca2+ and Na+ conductances.
Also both types of conductances have been shown to be activated in
dendrites by subthreshold EPSPs (Magee and Johnston
1995b
; Magee et al. 1995
; Schiller et al.
1997
; Stuart et al. 1997
). Therefore a
regenerative Na+ and/or
Ca2+ conductance is the likely mechanism
underlying supralinear summation.
The cases of supralinear summation that did not show a clear
regenerative hump might represent activation of dendritically located
persistent Na+ current (Schwindt and Crill
1995). Alternatively, a small regenerative response generated
distally might have been smoothed by dendritic filtering.
If activation of dendritic Na and/or Ca conductance caused supralinear summation, then why didn't larger EPSPs cause more supralinearity? One explanation is that the larger EPSPs, while activating more Na+ and Ca2+ conductances, will also activate K+ conductances that will counteract the boosting effects from Na+ and Ca2+ conductances. Additionally, in our experiments there is not necessarily a proportional relationship between EPSP size measured at the soma and EPSP size in the dendrites since we do not know the location along the dendrites of the synapses we activated and the location likely varied in different neurons. Indeed there was a suggestion that, for the more proximal evoked EPSPs, the larger ones resulted in a larger SR (SR increased in 4 of the 5 neurons where we systematically increased the strength of the Px stimulus). However, the relation of SR to EPSP size did not reach significance for the whole population of neurons (Fig. 5A).
Inwardly rectifying K+ current (GIRK) was
recently found to cause supralinear summation of EPSPs in a leach
neuron (Wessel et al. 1999). GIRK is present in the
dendrites of neocortical pyramidal neurons (Takigawa and
Alzheimer 1999
). GIRK would be expected to cause more
supralinear summation of EPSPs as membrane potential becomes
hyperpolarized. Therefore GIRK might also have contributed to the
supralinear summation that we observed. It is unknown if GIRK is
completely blocked by QX-314. A residual GIRK may account for summation
not becoming sublinear in QX-314-filled neurons.
Functional implications
What implications do our results have for information coding by
neocortical neurons? On average, EPSP summation was only slightly greater than linear. However, any model of synaptic integration must
take into account three results. First, we observed a wide range of SR
values (from linear to supralinear) that did not depend on differences
in the properties of individual neurons. Second, most of the
supralinear summation occurred during synaptic events separated by 5
ms. Finally, supralinear summation was unlikely unless the EPSPs were
preceded by membrane hyperpolarization.
Do the conditions that favored supralinear summation ever occur
normally? Based on dual recordings from pyramidal neurons with
patch-pipettes, hyperpolarizing the soma by 15 mV (e.g., from 70 to
85 mV) would cause the membrane potential of the apical dendrite (at
500 µm from the soma
a distance corresponding to halfway between
the Ds- and Px-stimulating electrodes) to hyperpolarize by
approximately equal to
4 mV (e.g., from
70 to
74 mV)
(Stuart and Spruston 1998
). Therefore 4 mV below resting
potential represents the maximum dendritic hyperpolarization (at 500 µm) in our experiments. Periods of membrane hyperpolarization to
approximately equal to
75 mV lasting several hundred milliseconds are
frequently observed during in vivo recordings from neocortex
(Stern et al. 1997
).
Supralinear summation of nearby inputs might favor coincidence
detection if the boosting is due to conductances with a rapid time
course (e.g., 1 ms) (Softky 1994
). Alternatively,
longer-lasting supralinear summation would favor temporal integration.
The cases of supralinear summation we observed were associated with a
regenerative response originating in the dendrite, but supralinear
summation occurred even when the two stimuli were separated by 5 ms.
Thus events occurring within an
5-ms time span would be temporally integrated and seen as coincident compared with events separated by
longer time intervals that summed linearly. Taken together, our results
suggest that supralinear summation of EPSPs would likely cause
synchronization and an initial boost to excitatory input after a period
of inhibition. However within a few milliseconds of depolarizing
activity, EPSPs would switch to linear summation. If linear summation
results from separated synapses, the supralinear summation must result
from closer synapses. But only linear summation is expected during
sustained depolarization. Thus during continuous activity in the
neocortical neurons it may be reasonable to ignore the spatial location
of synaptic inputs when considering their relative influence on each other.
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ACKNOWLEDGMENTS |
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We thank J.F.M van Brederode for helpful suggestions during the experiments, M. D. Binder and P. C. Schwindt for comments on the manuscript, R. Lee for technical assistance, and K. Bumsted, A. Hendrickson, and A. Erickson for assistance with histological procedures.
This work was supported by a VA Merit Review and National Institutes of Health Grant DC-02254 and Training Grant GM-07108.
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FOOTNOTES |
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Address for reprint requests: W. Spain, Neurology (127), VA Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108.
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 29 July 1999; accepted in final form 18 February 2000.
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REFERENCES |
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