Department of Anatomy and Neurobiology, St. Louis University, St. Louis, Missouri 63104
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ABSTRACT |
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Kogo, Naoki and Michael Ariel. Response attenuation during coincident afferent excitatory inputs. The linearity of the synaptic summation of two unitary excitatory synaptic events was investigated during whole cell recordings from retinal target neurons in an eye-attached isolated brain stem preparation. Pairs of unitary excitatory postsynaptic potentials (EPSPs) were evoked by bipolar stimulation electrodes that were directed to two distinct foci on the retinal surface based on the visual receptive field boundaries. The interval between stimulation of each retinal site was incremented by 0.5-1 ms to quantify the time course of nonlinear summation using an exponential fit. Response facilitation was never observed; however, the coincident arrival of synaptic inputs caused a response attenuation in 26 of the 37 pairs studied. Twelve of the 26 pairs had time constants of their attenuation that were similar to the time constants of the decaying phases of the first EPSPs of each pair. This suggests that the attenuation of these 12 pairs may be entirely due to voltage-dependent mechanisms, such as a reduction in driving force or a change of the activity of voltage-sensitive channels. On the other hand, the 14 other pairs had their time constant of attenuation shorter than the time constants of the decaying phase of the first EPSP. In fact, the attenuation time constants were often closer to the time constants of the decaying phases of the first excitatory postsynaptic currents of each pair. This finding suggests that the attenuation of these 14 pairs involve a shunting mechanism due to the opening of synaptic channels. The presence of this conductance-dependent mechanism is supported by the finding of asymmetric effects on the time course of attenuation when the stimulation sequence was reversed. These results are discussed in terms of the processing by neurons of coincident excitatory inputs onto spatially distinct points of their dendritic trees.
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INTRODUCTION |
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When two synaptic inputs coincide at different
synapses on a postsynaptic neuron's membrane, those inputs add to
produce a larger postsynaptic response. An intriguing question is
whether the summed response is simply the linear sum of the separate
responses. The possibility of nonlinear summation has been suggested
theoretically (Koch et al. 1983; Martiel et al.
1994
; Shepherd and Koch 1990
) and shown
experimentally using unitary synaptic inputs (Barrett and Crill
1974
; Burke 1967
; Haag et al.
1992
; Kuno and Miyahara 1969
; Lohmann and
Algur 1995
; McNaughton et al. 1981
;
Skydsgaard and Hounsgaard 1994
; however, see Cash
and Yuste 1998
; Jagadeesh et al. 1993
;
Langmoen and Andersen 1983
; Redman and Walmsley
1983
; Zhang et al. 1998
). Several mechanisms
have been suggested to account for nonlinear summation, including a
change in driving force at the synapse (Kuno and Miyahara
1969
), an activation or deactivation of voltage-sensitive
channels in the membranes of the dendrites and/or soma (Martiel
et al. 1994
; Shepherd and Brayton 1987
;
Shepherd et al. 1985
; Skydsgaard and Hounsgaard
1994
; Torre 1981
), and the conductance change
due to the opening of synaptic channels influencing the cable
properties of the dendrites (i.e., "shunting mechanism")
(Barrett and Crill 1974
; Burke 1967
;
Koch et al. 1983
; Qian 1990
;
Rall 1964
; Rall et al. 1967
).
Shunting mechanisms by inhibitory synaptic inputs have been shown to
have strong effects on the size of a coinciding excitatory postsynaptic
potential (EPSP) (Callaway et al. 1995; Cauller
and Connors 1994
; Kapur et al. 1997a
;
Langmoen and Andersen 1983
; Rall et al.
1967
). It also was indicated that this nonlinear interaction can happen between "calculated" unitary EPSPs based on the
membrane properties and neuronal geometry of cat motoneurons
(Barrett and Crill 1974
; Shepherd and Koch
1990
). Unlike the first two voltage-dependent mechanisms for
nonlinear summation (changes in driving force and voltage-sensitive
channels), the shunting mechanism is unique because it is based solely
on the dendritic membrane's conductance change, irrespective of the
membrane voltage. Also because one region of synaptic membrane
"siphons" current from an input signal as it travels along the
dendrite, it also is expected that the shunting mechanism will depend
on the relative locations of both of the synapses that are being
activated (Koch et al. 1983
; Miles et al.
1996
; Qian et al. 1990
). An analysis of the time
course of the nonlinear summation might reveal these characteristics of
the shunting mechanism experimentally. One expects that the time course
of conductance change at a synapse to be faster than that of the
membrane potential change because the postsynaptic potential (PSP) is
the temporal integral of the input current (Barrett and Crill
1974
; Kapur et al. 1997a
,b
; zharvPearce
1993
). One can test whether the time course of any nonlinearity
in the summation is similar to, or faster than, the time course of the PSP to reveal temporal difference between voltage-sensitive mechanisms and shunting mechanisms.
It has been reported that shunting that evokes a nonlinear summation
should be more effective when the siphoning of current occurs at
proximal site on a dendrite relative to the other synaptic input on a
more distal part of the same dendrite (Koch et al. 1983;
Skydsgaard and Hounsgaard 1994
). When two coinciding
EPSPs are considered, as in this report, an asymmetric interaction
between proximal and distal EPSPs may be present. However, when two
EPSPs are evoked so that both peaks coincide exactly, the membrane
response is a summation of both synaptic events that are
affecting each other nonlinearly. Thus the measured nonlinearity cannot
be attributed solely to the proximal and distal EPSP. A protocol
therefore was developed to study asymmetries that may be dependent on
synaptic location as revealed by the time course of the nonlinearity in the summation. Such details of the nonlinear summation, although not
presented in other reports of nonlinear summation (Burke
1967
; Haag et al. 1992
; Kuno and Miyahara
1969
; Lohmann and Algur 1995
; McNaughton
et al. 1981
; Skydsgaard and Hounsgaard 1994
),
would give some clue as to the mechanism(s) underlying the nonlinearity.
We recently reported unitary synaptic events during whole cell
recordings of cells in the basal optic nucleus (BON) of the turtle
brain in vitro (Kogo and Ariel 1997). The BON is an
accessory optic nucleus that sums inputs from the contralateral retina
to create a direction-sensitive retinal slip signal for the oculomotor system. Unitary EPSPs from retinal ganglion cells can be evoked efficiently in BON cells by microstimulation of the retinal surface based on the BON cells receptive field. We, therefore, decided to use
this experimental model to explore the properties of EPSP interactions.
We report here the sublinear summation of these unitary EPSPs and the
results of time course analysis of this phenomenon.
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METHODS |
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In vitro preparation
The turtle eye-brain preparation was described in detail
elsewhere (Kogo and Ariel 1997). After >1 h in ice, the
entire brain of a turtle, Pseudemys scripta elegans, was
removed with the eyes attached. The telencephalon was removed within 15 min of decapitation, preventing conscious sensations before the tissue
equilibrates to room temperature. The spinal cord and medulla were next
removed, the eyes were hemisected, and the preparation was placed
ventral side up in the superfusion chamber. The ionic composition of
the superfusate was (in mM) 130 Na, 2.0 K, 3.0 Ca, 2.0 Mg, and 97 Cl,
bubbled with 95% O2-5% CO2 (pH 7.6). In 10 brains, the dorsal structures of the brain stem, i.e., dorsal portion
of thalamus, optic tecta, and cerebellum, also were removed. Inhibitory
postsynaptic potentials (IPSPs) rarely were observed, and bicuculline
was not added to the superfusate. In the other five brains, the dorsal structures were kept intact and BON cells often showed spontaneous IPSPs. In those five brains, 100 µM of bicuculline was added to the
superfusate in the brain chamber and the IPSPs were blocked. The
properties of EPSP summation in these two preparations were similar.
Recordings, stimulations, and experimental protocol
In situ recordings were made using patch electrodes in a
standard whole cell configuration (Blanton et al. 1989).
The pipette solution was (in mM) 114 KMeSO4, 2.3 CaCl2, 1.2 MgCl2, 10.0 HEPES, 5.0 EGTA, and 2.0 ATP (pH 7.3-7.4). Data were only used if the series resistance did not
change throughout the cell's recording. In 13 cases, the pipette
solution was modified by replacing 10 mM of KMeSO4 by same
concentration of a lidocaine derivative, QX314, which presumably
enhanced the space clamp of the recording. Although there are several
effects of QX314 on spike responses and Ih
(Perkins and Wong 1995
), nonlinear summation of EPSPs
appears unaffected by this drug.
To evoke a pair of EPSPs in BON cells (Ra and
Rb), two bipolar stimulation electrodes were placed on the
retinal surface at two distant sites (Sa and
Sb) within the receptive field, as guided by the visual
responses of that BON cell. The distance between the two poles of each
bipolar electrode was ~25 µm. Unitary EPSPs (synaptic responses due
to an input from a single retinal ganglion cell) were evoked from those
sites by adjusting the position of each stimulation electrode so that
very small stimulus currents into the retina would evoke a response in
the BON cell of a short latency (5.3 ms ± 1.1, n = 59) (measured by Kogo and Ariel 1997). These evoked
EPSPs were considered unitary when the response was small at a clear
stimulus threshold and when much higher stimulus current was required
before the response amplitude would increase, presumably due to current
spread that would recruit inputs from distant retinal ganglion cells
(see Kogo and Ariel 1997
).
To quantify the interaction between two unitary synaptic responses, the interval between Sa and Sb stimulation first was adjusted so that Ra and Rb approximately coincided. Then relative to that interval of coincident responses, stimulus intervals were increased (in either 0.5- or 1.0-ms increments) to generate a set of 20 different intervals. This whole procedure was repeated 100 times with the interval between these 2,000 paired stimulations separated by 0.4 s. In 19 cases, same-site paired stimulation also was performed as a control.
Measurement of nonlinearity
During the off-line analysis, each response first was examined and then averaged for each tested interval. Then the peak latency (tpb) and peak amplitude (PbCTRL) were measured for the Rb response alone. The difference between the summed response and Ra was measured at a time tpb later from Sb stimulation for each tested interval. Finally this difference was plotted as an attenuation plot and compared with the control peak amplitude (PbCTRL).
ATTENUATION PLOT.
To quantify whether the summed response (Rsum) differed from the
mathematical sum of Ra and Rb, a function,
P, was defined as follows. First, Ra and Rb
were evoked separately (RaCTRL and RbCTRL).
Their peak amplitudes and latencies were measured and designated as
PaCTRL, PbCTRL, tpa, and
tpb, respectively. Rsum and Ra were, measured
at a time tpb ± 100 µs after the second stimulation. The
difference of the averaged values of Rsum and Ra is defined
as
P.
P was plotted as a function of the
interresponse peak interval (IPI) on the abscissa, defined as
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EXPONENTIAL FIT.
P appeared to increase exponentially toward
PbCTRL. Therefore
P, as a function of IPI,
was fit to
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ATTENUATION INDEX AND TIME CONSTANT.
An attenuation index, AI, was computed for each fit as a percent ratio
of M to a mathematical sum of RaCTRL and
RbCTRL
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RESULTS |
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Two retinal unitary EPSPs first were evoked separately during a whole cell recording of a BON cell (Fig. 1A). The BON cell's receptive field was plotted based on small visual patterns drifting in the preferred direction. Then each stimulating electrode was positioned on the retinal surface at different edges of that receptive field. Finally each electrode's location is adjusted further slightly so that minimal levels of stimulus current would evoke unitary excitatory synaptic events from each retinal site.
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An example of a pair of control unitary synaptic responses of the same
cell is shown in Fig. 1B. They were evoked by stimulating two distant sites close to the border of the receptive field as shown
in Fig. 1C. The stimulation sites were separated to assure that the summation measured was from different afferent inputs. In Fig.
1D, the receptive field map is superimposed on a retinal whole-mount drawing of ganglion cells backfilled by a tracer injection into the contralateral BON (kindly provided by Dr. Eldred, Boston University, Boston, MA). Surely, the ganglion cells specifically stimulated at Sa and Sb cannot be determined
from anatomic material of another retina. However, it is clear that
within the receptive field, ganglion cells are widely spaced. Moreover,
adjacent ganglion cells probably do not project to the same BON cell
(see Kogo et al. 1998). It is therefore likely that
distant stimulation sites will stimulate separate ganglion cell bodies
or their axons as they project toward the optic disk. The unitary
inputs to the BON cell also were known to be from different afferents
because they had different latencies, different waveform shapes, and
different amplitudes.
To evaluate the synaptic summation of two different unitary inputs described in the following text, some basic properties of single unitary EPSPs first were studied (Fig. 2). Figure 2A shows the difference of the time course of an EPSP and an EPSC by inverting and scaling the EPSC amplitude to match the EPSP (Ra as shown in Fig. 1). As expected, the time course of the EPSC is much shorter than that of the EPSP, i.e., a shorter peak latency (tpEPSC) than that of the EPSP (tpEPSP) and also a faster decaying phase for the EPSC.
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Because membrane depolarization reduces the driving force of an EPSP,
changes in membrane potential had to be considered as a factor causing
nonlinear summation of EPSPs. In seven unitary EPSPs, the effect of
membrane potential on the EPSP amplitude was measured as shown in Fig.
2B. A unitary EPSP response was evoked from the retina while
the postsynaptic cell was adjusted to different membrane potentials by
current injection through the recording pipette. In the example shown,
the EPSP size decreased only slightly as a function of membrane
potential (0.058 mV per mV of depolarization). On average, the ratio
of the EPSP amplitude change to membrane potential change was
0.0149 ± 0.0043 mV/mV (n = 7). Therefore it may
be improbable that attenuation of an EPSP is due exclusively to another
EPSP's voltage response of the size measured at the recording pipette.
If the same ganglion cell or its axon was to be stimulated at two
stimulating sites in the retina, the synaptic response in the BON may
show a refractory period of the two action potentials as they travel in
the optic nerve to the BON. Therefore the refractory period of a single
unitary EPSP also was measured to demonstrate that it appears different
from the nonlinear summation observed using two different unitary EPSPs
(Fig. 2C compared with Fig. 3A). A refractory period was
observed by stimulating a same retinal site twice, employing the same
stimulation protocol that quantifies the interaction of two unitary
EPSPs (see METHODS). Using an interstimulus interval
increment of 1.0 ms, Fig. 2C shows that the first three stimulus pairs failed to evoke the second response (absolute refractory period of <3 ms), the fourth stimulus pair evoked the second response occasionally (relative refractory period), and stimulus pairs with
intervals 4 ms always evoked the second response. This pattern of
short interval response failure also would occur if two sites of
unitary retinal stimulation did in fact stimulate the same unitary EPSP
in the postsynaptic cell.
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This artifactual form of nonlinear summation (spike refractory period
during paired stimulation of the same afferent) was avoided by
separating the two retinal stimulation electrodes and by using unitary
EPSPs of different waveform shapes and amplitudes. After examining the
control responses of single unitary synaptic events, interactions of
two afferent inputs to the BON were measured during small interresponse
peak intervals (Fig. 3, A and B, current- and
voltage-clamp recordings, respectively). Specifically, the amplitude of
the summed response was measured at a time after the second stimulus
that corresponded to the peak latency of the second control response
(Fig. 3C). That amplitude then was compared with the
amplitude of the first control response at that same point in time (see
METHODS). This difference (P) then was
plotted (Fig. 3, A and B, insets) for each
tested interval to determine how synaptic summation changed as a
function of the interval between synaptic events.
Interactions of evoked responses (37 pairs recorded in current-clamp mode, 23 of which also recorded in voltage-clamp mode) were analyzed. We found that when two synaptic responses are nearly coincident, they often summed sublinearly (compare the left vertical arrow to the right vertical arrow in Fig. 3C). This attenuation phenomenon was not a result of GABA inhibition because bicuculline was added to the superfusate when using whole brain stem preparations (in which inhibitory pathways were still intact) and the phenomenon was still observed.
The results of the quantification of the nonlinear synaptic
summation are presented graphically in Fig. 3, A and
B, right. Specifically, when the two responses strongly
overlapped in time (the interresponse peak interval is near 0), the
summed response minus the first response was often much smaller than
the control second response (i.e., P < PbCTRL). As the interval between the events increased, the
amplitude of the second response's waveform increased toward the value
of the control response to the second stimulus. The two extreme results
of these experiments are shown in Fig. 4
(A: strong attenuation; B: close to linear
summation). Facilitation of the summation never was observed in BON
cells. Because attenuation appeared to decay exponentially as the
stimulus interval increased, the data from each experimental set were
fit by computer to an exponential function to compute an attenuation index (AI) and its time constant (
attn). AI represents
our estimate of the amount of response attenuation when the peaks of
both responses perfectly coincide, as a percentage of the sum of two
control peak amplitudes, PaCTRL and PbCTRL.
Histograms of AI and
attn for all stimulus pairs are
shown in Fig. 4, C and D, respectively. Because
of the response variability, attenuation of synaptic summation appeared
to the experimenter as a clear phenomenon when the AI value was larger
than 5. Most of the stimulus pairs showed some degree of attenuation
(Fig. 4C). When the AI value was <5, the
attn was often >100 ms; Fig. 4D, rightmost
bin.
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In 26 experimental data sets that showed attenuation in the
current-clamp mode (AI > 5 and attn < 100), the
time constant of the attenuation (
attn) was compared
with the time constant of the decaying phase of Ra in the
current-clamp mode (
R).
attn was either
close to
R (Fig.
5A) or shorter than
R (Fig. 5B). The results for these two time
constants are plotted in Fig. 5C. Twelve data sets had
similar time constants for attenuation and Ra (
), while
14 sets had shorter time constants for attenuation (
). These
findings suggest that although attenuation may be due to a
voltage-dependent mechanism in some cases, a conductance-dependent mechanism also may play a role in this attenuation phenomenon.
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Among the 14 data sets that showed shorter attn than
R in the current-clamp mode (RaCC), 7 of
them also had Ra recordings made in the voltage-clamp mode
(RaVC). These seven sets were chosen to compare
attn to
R in these two recording modes.
Their
attn values were closer to their corresponding
RaVC time constants than to their corresponding
RaCC time constants (Fig. 5D). The fact that
attn values were in between the time constants of
RaCC and RaVC in some cases (e.g., Fig.
5E), may suggest that this attenuation phenomenon can
involve both voltage- and conductance-dependent mechanisms.
The asymmetry of the time course of attenuation also was investigated
to further support the role of a shunting mechanism in this sublinear
summation phenomenon. This analysis was performed on the assumption
that asymmetric response differences for different sequences of
stimulation would correlate with response differences to different
synaptic locations on the dendritic tree. As shown in Fig.
6A, an EPSP response
Ra was evoked at a fixed time while Rb was
evoked at different intervals later. In Fig. 6B, on the other hand, Rb was evoked first and Ra was
evoked later. Reversing the sequence did not change AI significantly in
all samples (Fig. 6D). However, the time courses of the
attenuation of these opposite sequences were often different (see Fig.
6C in which P was normalized, based on the
example shown in Fig. 6, A and B).
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The attn values of the opposite sequences then were
compared with the time constants of the first responses
(
R) in these sequences, as shown in Fig. 6E
(8 pairs, 16 data points). The data points with open symbols indicate
that
attn was similar to
R (tentative
voltage-dependent mechanism). The data points with filled symbols
indicate that
attn was shorter than
R
(tentative conductance-dependent mechanism). On the basis of those
results, the eight pairs were categorized into two groups. The first
group (n = 3, circles) showed the voltage-dependent
attenuation in both sequences. The second group (n = 5, squares) showed the conductance-dependent attenuation in at least one
sequence. In four of these five pairs, the opposite sequence had
attn similar to
R (open squares). In the
one pair of retinal inputs for which
attn was shorter than
R in both sequences (asterisk), coincident
responses of that pair also showed the strongest attenuation index of
the entire data set of these experiments (see rightmost bin of Fig.
4C). This suggests that their synapses were close enough to
each other on the BON dendritic membrane to produce a shunting effect
in either sequence.
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DISCUSSION |
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Pairs of unitary EPSPs from two retinal locations were evoked in BON cells to study the linearity of the synaptic summation. In many cases, a clear attenuation was observed, whereas response facilitation never occurred. By analyzing the time course of this nonlinear summation, possible mechanisms that underlie this phenomenon were investigated.
Evoking unitary EPSPs in BON cells
The "minimal stimulation" method has been used commonly to
evoke unitary excitatory synaptic events in cortical neurons in brain
slice preparations where the stimulating electrodes were placed in
either white matter or areas densely populated with neurons. At those
stimulation sites, the current was reduced to a minimum so that unitary
postsynaptic responses would be evoked (Isaac et al.
1995; Liao et al. 1995
; Malinow and Tsien
1990
; Raastad 1995
; Stevens and Wang
1994
). In our experimental model, direction-sensitive retinal
ganglion cells that project to the BON were activated by bipolar
electrodes for which the distance between each pole was about the same
size as the ganglion cell soma (~25 µm). These ganglion cells are
known to have the largest cell bodies in the turtle retina and are
diffusely scattered across the retinal surface (Reiner
1981
; Zhang and Eldred 1994
). In BON, the
amplitudes of unitary EPSPs from these retinal inputs are very large
(7.8 ± 5.2 mV) (Kogo and Ariel 1997
) and come from afferents separated by as much as 4 mm on the retinal surface. These
features in this vertebrate brain preparation increase the likelihood
that the "minimal stimulation" method will evoke unitary synaptic
responses, as opposed to responses of an aggregate of afferent inputs.
Thus a pair of retinal unitary EPSPs could be evoked readily within the
duration of a stable whole cell recording of a BON cell.
Unitary EPSPs in BON cells have been shown to be monosynaptic
(Kogo and Ariel 1997). The onset latencies for these
EPSPs were quite constant for repeated stimulations, using a range of
stimulus currents that remained below the level that recruited other
EPSPs. Also it was shown that during short-interval paired stimulation of a single retinal site, the unitary EPSP followed each pulse of the
pair if the interpulse interval was >3.5 ms (± 2.0, n = 16). The second response failed when the interval was shortened, suggesting that the responses were monosynaptic. These responses also
followed high-frequency repetitive stimulation without a change in
waveform shape, indicating that nonmonosynaptic or nonunitary inputs
did not contribute to the unitary response (Kogo and Ariel 1997
).
The conclusions made here about EPSP summation are based on the assumption that the retinal stimulation truly evoked unitary independent EPSPs. Even without that assumption, it is still unlikely that nonlinear summation would result from interaction at the retinal level by activating a common amacrine cell input. Figure 1D shows the density of ganglion cell inputs to the BON. Between the two stimulation sites are many intervening retinal ganglion cells that project to the BON. Not shown are also thousands of unlabeled amacrine cells that perform local processing for each ganglion cell. It is therefore unlikely that the different sites of stimulation would evoke a common amacrine cell input to distant ganglion cells.
It is also unlikely that the two sites affected the same ganglion cell,
e.g., one site at its soma and the other site elsewhere along its axon
as the ganglion cell projected to the optic disk. As seen in Fig.
1D, the two stimulation sites were not in line with the
direct path between the soma and the optic disk. If the same ganglion
cell would have been excited by the two stimulating electrodes, one
also would expect that the second response would suddenly appear after
the interstimulus interval exceeded the absolute refractory period (3.5 ms ± 2.0, measured by same-site paired pulse stimulation)
(Kogo and Ariel 1997). However, unlike the
"all-or-none" response observed in same-site paired stimulation experiment (Fig. 2C), two different sites evoked two
distinct responses that were observed in all 100 response traces at all interstimulus intervals. Also, the observed response attenuation did
not occur in an all-or-none manner but decreased exponentially as that
interval between the two different sites increased.
Mechanisms of nonlinear summation
Three potential mechanisms for attenuation were considered: a decrease in the driving force at one synapse due to the depolarizing effects of another synapse, a change in the activities of voltage-sensitive channels at one synapse due to the depolarizing effects of another synapse, and a change in membrane conductance at one synapse due to the opening of another synapse's channels that siphon off the input current.
A change in driving force may be an unlikely mechanism of attenuation because, in most cells, the depolarization produced by Ra was only 5-10 mV, whereas the potential dependency of the EPSP amplitudes was 0.015 mV/mV (presumably measured at the soma). However, the amplitude of an EPSP at the synaptic membrane is expected to be larger than that recorded elsewhere on the membrane. If so, a large depolarization potentially could cause a strong attenuation because of a change in the driving force on the charge carriers of the synaptic current.
If attenuation is due to the second mechanism, voltage-sensitive
outward currents are activated during membrane depolarization. Thus a
stronger depolarization during the summed EPSP, when compared with that
of a single EPSP, would activate more currents and attenuate the summed
potentials. If, however, either voltage-sensitive inward currents are
activated or inward rectifiers are deactivated, the result should be a
facilitation of the summed signals. A hyperpolarization-activated current, Ih, which is present in most BON cells
(Kogo and Ariel 1997), also might contribute to
attenuation by causing a weaker rebound excitation at the second
response. A role for Ih is unlikely because
attenuation was observed at membrane potentials above
60 mV (above
the threshold of Ih) (Kogo and Ariel
1997
) and in several recordings made with patch pipettes
containing QX314 (shown to block Ih)
(Perkins and Wong 1995
). We also can exclude a role for
voltage-sensitive N-methyl-D-aspartate (NMDA)
receptors in the nonlinearity because the retino-BON synapse appears
mediated solely by AMPA receptors (unpublished observations).
It also was observed that the interresponse peak interval that showed the strongest attenuation did not necessarily correspond to the time of the highest peak amplitudes of the summed response (see Fig. 4A). Therefore these voltage-sensitive mechanisms, i.e., changes in driving force at the synaptic membrane or in the activation of voltage-sensitive channels, are not sufficient to explain all of the observed aspects of nonlinear summation. In addition, the time course of the attenuation did not always correspond to the voltage change by Ra as described in the following text. Therefore a mechanism in which synaptic channel openings caused a conductance increase in the dendritic membrane was considered. This third mechanism is similar to the shunting effect that GABAergic synaptic channels have on excitatory synaptic inputs. In both cases, synaptic channels open, thereby "siphoning" an excitatory signal and attenuating its response. Also both shunting mechanisms are very sensitive to the arrangement of the pair of synapses on the dendritic tree.
Time course analysis
If attenuation is due to either voltage-sensitive channel activations or driving force changes (voltage-dependent mechanisms), the time course of the attenuation should follow the time course of the voltage change. If, on the other hand, attenuation resulted from the shunting of synaptic current by the opening of other synaptic channels (conductance-dependent mechanism), this attenuation should be shorter than the voltage change reflecting the time course of the synaptic conductance change. Therefore the time course of the attenuation may be useful to distinguish the conductance-dependent mechanism from voltage-dependent mechanisms of the attenuation.
The time course of EPSC reflects the time course of the conductance
change for a perfect space-clamp condition because an EPSC is a product
of the conductance times the driving force at a synapse. In theory,
therefore, comparing the time course of the attenuation with EPSC of
Ra may give some insight in the participation of the
conductance-dependent mechanism in the attenuation. However, the BON
recordings probably were made near the cell body and may not reflect
the time course of the original input waveform at the synapse because
of filtering by the cable properties of the dendritic tree (Rall
1977). Even though a perfect space clamp may not have been
achieved in the voltage-clamp mode, the time course of the EPSC is much
shorter than that of the EPSP (Fig. 2A), indicating that the
relative time course between EPSC and EPSP at the synapse may be
maintained at the recording site.
To correlate the short time course of the attenuation with that of a
synaptic conductance change, the time constants of P were
compared with that of the EPSPs and EPSCs. The result indicated that
there were two groups of data points (Fig. 5C). One group showed a time constant of
P that was shorter than that of
the EPSP, whereas in the other group, both time constants of
P and EPSP were similar. Each of the pairs in the first
group of data points had a
P time constant closer to the
time constant of their EPSC than that of their EPSP (Fig.
5D). This suggests that the synaptic conductance change
during some of the retinal input pairs can indeed cause a strong
interaction between the input signals.
The nonlinear synaptic summation observed here also may be reflected in
relationship between the synaptic inputs and the spike responses of the
BON cells. We have reported previously that BON cell's spike frequency
was related linearly across a wide range to the level of current
injected through the patch pipette (Kogo and Ariel
1997). Assuming that the spike frequency also is related linearly to the net synaptic current, although this summed synaptic signal is related nonlinearly to the coinciding inputs, one would expect that spike frequency response would be sublinear as well during
simultaneous synaptic inputs.
Dependency of the time course to sequence of paired stimulation
Different pairs of unitary EPSP(C)s showed different amounts of nonlinear summation (Fig. 4C). These differences may relate to the electrotonic distance of each pair of synapses on the dendritic tree. In theory, if two retinal afferents make synapses that are close to each other, then their inputs would influence a common dendritic area to produce a large amount of voltage change that might cause this nonlinear interaction. Because each set of stimulated afferents was selected at random, the distribution of the values of the attenuation may reflect the variety in the relative locations of the two synapses.
The locations of synapses is an even more crucial factor for the conductance-dependent mechanism. The effectiveness of the conductance change of an earlier synaptic event on a later event would depend on the location of the earlier synapse relative to the dendritic pathway by which the later synaptic signal conducts toward the cell body. The effectiveness of the conductance change at the first synapse would be strong if it is located on the proximal site and the second synapse on the distal site of the same (or nearby) dendrite. This suggests a possible asymmetry of the conductance-dependent EPSP interaction in terms of the sequence of occurrence of the proximal and the distal synaptic events.
By reversing the sequence of the paired stimulation, we investigated whether this asymmetry can be observed in the parameters of nonlinear summation, i.e., attenuation index and time constant. As explained in METHODS, AI is the ratio of the attenuation at time 0 to the total peak amplitude of RaCTRL and RbCTRL. Because time 0 is defined as when two peaks of the responses coincide, it is not surprising that the AI numbers from the two opposite sequences were quite similar (Fig. 6D).
Time constants, on the other hand, were affected dramatically by
reversing the sequences. A group of data sets (Fig. 6E,
squares) showed the conductance-dependent attenuation in at least one
sequence (namely, attn is shorter than
R). In four of these five pairs, the opposite sequence
showed "voltage-dependent" attenuation (
attn is
not statistically different from
R). This suggests that,
in the latter sequence, a conductance-dependent mechanism was no longer
a dominant factor causing attenuation. In another words, one of the two
synaptic events had a strong "unidirectional" effect to the other
synaptic response through the conductance change. This presumably
reflects the location dependent asymmetrical influence of the synaptic
conductance change.
Nonlinear synaptic summation in other neural systems
A recent report indicated that in cultured pyramidal cells, the
summation of the EPSPs is quite linear (Cash and Yuste
1998). In their report, it was indicated that when NMDA
blockers were applied, the system showed sublinear summation,
presumably due to the lack of a compensatory voltage sensitivity
mediated by NMDA receptors. It is also possible that other
voltage-sensitive inward currents exist on the dendrite of
pyramidal cells. This suggests that these neurons have
"boosting" mechanisms to avoid the attenuation of the signals. In
more passive neurons, on the other hand, without such voltage-sensitive
elements located effectively in appropriate cellular structures, the
summation may occur in the nonlinear fashion. This may very likely be
the reason that the occurrence of the nonlinear summation depended on
the neurons studied in the previous reports. The electrotonic isolation
of synapses by spines (Shepherd et al. 1985
) and active
channels on spines (Miller et al. 1985
; Segev and
Rall 1988
) also would help maintain linearity of the signal.
BON cells, on the other hand, seem not to have spines on dendrites, and
their retinal EPSPs are mediated by AMPA receptors only (unpublished
observations). Without the morphological isolation of individual inputs
on spines or the presence of voltage-sensitive boosting mechanisms to
compensate for the attenuation of input signals, BON cells display the
fundamental properties of nonlinear interactions between synaptic
inputs. In conclusion, our experiments that use the retinal unitary
inputs to BON cells revealed basic properties of the EPSP interactions presumably due to both voltage- and conductance-dependent mechanisms that may operate in other neural systems as well.
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ACKNOWLEDGMENTS |
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We thank Drs. J. Huettner, N. Spruston, and J. H. Steinbach for helpful comments.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-33190 (to M. Ariel).
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FOOTNOTES |
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Address for reprint requests: M. Ariel, Dept. of Anatomy and Neurobiology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104.
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 6 April 1998; accepted in final form 24 February 1999.
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REFERENCES |
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