Response Attenuation During Coincident Afferent Excitatory Inputs

Naoki Kogo and Michael Ariel

Department of Anatomy and Neurobiology, St. Louis University, St. Louis, Missouri 63104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, Delta 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 Delta P. Delta P was plotted as a function of the interresponse peak interval (IPI) on the abscissa, defined as
<IT>IPI=tp<SUB>b</SUB>−tp<SUB>a</SUB>±&Dgr;</IT><IT>S</IT>
where Delta S is a stimulus interval. Thus when IPI = 0, the two response peaks coincide.

EXPONENTIAL FIT. Delta P appeared to increase exponentially toward PbCTRL. Therefore Delta P, as a function of IPI, was fit to
<IT>&Dgr;</IT><IT>P</IT><IT>=P<SUB>bCTRL</SUB>−</IT><IT>M</IT><IT>∗exp</IT>(−IPI<IT>&cjs0823;  &tgr;</IT><SUB><IT>attn</IT></SUB>)
This exponential fit was performed by SPSS statistical analysis program (SPSS, Chicago, IL) which estimated the parameters M, the y intercept, and the time constant tau attn. It also gave the standard error of the parameters used later for the statistical analysis. A curve of each fit, based on M and tau attn, was plotted along with data points of Delta P as shown in attenuation plots (Delta P vs. IPI).

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
<IT>AI=100∗</IT><IT>M</IT><IT>&cjs0823;  </IT>(<IT>R<SUB>aCTRL</SUB>+R</IT><SUB><IT>bCTRL</IT></SUB>)
Because M is a difference between RbCTRL and y intercept of the exponential curve, AI is the estimated value of the attenuation when the peaks of two responses coincide (at IPI = 0). AI may be an overestimation of the actual attenuation because the exponential curve occasionally went below Delta P at IPI = 0.

Time constants of EPSP and excitatory postsynaptic current (EPSC) were measured by a single exponential fit of the decaying phase along the same part of the signal where Delta P was measured. All statistical analyses used a Student's t-test (P = 0.5) performed by the SPSS statistics program.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. A: drawings of the experimenter's view of the turtle eye-brain preparation, ventral side up (ipsilateral eyecup not shown). Positions of 2 retinal landmarks are noted: the optic disk (denoted with a small solid black oval) and the visual streak (appearing as a curve within the eyecup). a: checkerboard pattern is projected onto the retina and then moved in 12 directions to determine the preferred direction of the basal optic nucleus (BON) cell. b: light spot is moved in the preferred direction to detect the edges of the BON cell's receptive field (circle within the eyecup). The spot then is placed on a receptive field edge to guide a bipolar stimulating electrode to the retinal surface (Sa). Spot is moved to another edge to guide a second bipolar stimulating electrode (Sb). Final position and stimulus strength of each electrode are adjusted to evoke unitary excitatory postsynaptic potentials (EPSPs) in the BON cell (Ra and Rb). B: representative voltage tracings of control responses Ra and Rb evoked separately (RaCTRL and RbCTRL) from 2 retinal locations. Each trace is an average of 100 current-clamp recordings. Peak latencies of RaCTRL and RbCTRL are indicated as tpa and tpb. C: specific retinal stimulus locations (Sa and Sb) on the edges of the BON cell's receptive field (partial circle) used for the example shown in B. N and T, nasal and temporal sides of the horizontal visual streak of the right eye; OD, optic disk. D: specific receptive field map shown in C is superimposed on a drawing of ganglion cell inputs to the BON. Two lines are added to suggest axonal paths to the optic disk from ganglion cells at Sa and Sb.

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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Fig. 2. A: superimposed current- and voltage-clamp traces, from the same cell shown in Fig. 1 during stimulation at Sa. B: traces of unitary EPSPs measured at 9 membrane potentials (average of 20 traces); displayed horizontally at half the time scale (6 ms, compared with 3 ms for the figure's other traces). Right: least squares linear regression of the EPSP amplitude as a function of the preceding membrane potential. C: responses to double shocks at a single retinal location (average of 100 traces). Arrowheads point up to the stimulation artifacts of the 2nd stimulation of each stimulus pair (20 different interstimulus intervals beginning with 0 ms and incremented by 1 ms); arrows point down to EPSPs that respond to the 2nd stimulus. Examination of the individual traces (not shown) revealed that 0/100 s-responses occurred during the 1st 3 pairs (intervals of 0-2 ms within the absolute refractory period), 55/100-s responses occurred during the 4th pair (3-ms interval within the relative refractory period) and 100/100-s responses occurred during the 5th-20th stimulus pairs.

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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Fig. 3. Experimental data set, using the example shown in Fig. 1. Averaged traces are overlapped and all aligned to Sa. Attenuation plots are shown to the right. A: current-clamp recordings using a 1-ms increment of the stimulus interval between Sa and Sb. For the shortest interval between Ra and Rb, the difference between the summed response (Rsum) and Ra was significantly smaller than RbCTRL. B: voltage-clamp recordings shown on the same time scale as in A using a 0.5-ms increment of the stimulus interval. C: 2 examples of the traces shown in A in 2 different stimulus intervals, superimposed with RaCTRL. Note that tps, the 2nd peak of the summed excitatory postsynaptic current (EPSC), occurred just before tpb. Measurement of the response was made at tpb ± 100 µs.

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 (Delta 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., Delta 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 (tau 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 tau 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 tau attn was often >100 ms; Fig. 4D, rightmost bin.



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Fig. 4. Two other examples of experimental data sets (current-clamp recordings) that showed a strong attenuation (A) and no attenuation (B). C and D: histograms of the attenuation indices (AI) and the time constants (tau attn) from all experimental data sets respectively (, 37 in current clamp; , 23 in voltage clamp). Note that as shown in B, when no attenuation was observed, the exponential fit resulted in a large time constant. Those data points are plotted together as >100 ms in D.

In 26 experimental data sets that showed attenuation in the current-clamp mode (AI > 5 and tau attn < 100), the time constant of the attenuation (tau attn) was compared with the time constant of the decaying phase of Ra in the current-clamp mode (tau R). tau attn was either close to tau R (Fig. 5A) or shorter than tau 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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Fig. 5. Two examples of a comparison of the time constant of the attenuation (tau attn) with that of Ra (tau R). A: tau attn and tau R were similar in this example. B: tau attn was shorter than tau R. C: scatterplot showing tau attn and tau R of all stimulus pairs (n = 26) chosen for the time course analysis. Straight line shows when both time constants were equal. Filled squares indicate the data points with shorter tau attn than tau R, whereas open squares indicate no significant difference between them (P > 0.5). D: tau attn values are compared (n = 7) with tau R in current clamp (square) and voltage clamp (triangle). tau attn values were either close to time constant in voltage clamp or in between the time constants of the two recording modes. E: example of response traces of a BON cell, showing the tau R of an EPSC (10 ms), the tau R of an EPSP (35 ms), and the tau attn of an attenuation plot (20 ms).

Among the 14 data sets that showed shorter tau attn than tau 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 tau attn to tau R in these two recording modes. Their tau attn values were closer to their corresponding RaVC time constants than to their corresponding RaCC time constants (Fig. 5D). The fact that tau 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 Delta P was normalized, based on the example shown in Fig. 6, A and B).



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Fig. 6. Effect of reversing the stimulus sequence on the nonlinear summation. A: response Ra was evoked at a fixed time while the other response Rb was evoked at different intervals later. B: Rb was evoked 1st and Ra was evoked later. C: time courses of the attenuation in the opposite sequences measured from the recordings shown in A and B. Note that the ordinate values were normalized by the control amplitude of the second response of each sequence. D: entire sample of pairs of attenuation indices obtained from opposite stimulus sequences. Straight line indicates where AI values from each sequence are equal. In all cases, AI values were not significantly different (P > 0.5). E: tau attn and tau R in these sequences are plotted (see Fig. 5C). Opposite sequences of same stimulus pair are connected by straight lines. Squares indicate the pairs that had a tau attn shorter than tau R in at least 1 of the 2 sequences. Open symbols indicate tau attn and tau R were not significantly different in these data points (P > 0.5). Note that in 4 of 5 pairs with shorter tau attn than tau R, reversing the sequence resulted in the tau attn to be closer to tau R, except 1 pair designated with asterisk. Circles denote stimulus pairs in which neither sequence had tau attn shorter than tau R.

The tau attn values of the opposite sequences then were compared with the time constants of the first responses (tau R) in these sequences, as shown in Fig. 6E (8 pairs, 16 data points). The data points with open symbols indicate that tau attn was similar to tau R (tentative voltage-dependent mechanism). The data points with filled symbols indicate that tau attn was shorter than tau 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 tau attn similar to tau R (open squares). In the one pair of retinal inputs for which tau attn was shorter than tau 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta P that was shorter than that of the EPSP, whereas in the other group, both time constants of Delta P and EPSP were similar. Each of the pairs in the first group of data points had a Delta 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, tau attn is shorter than tau R). In four of these five pairs, the opposite sequence showed "voltage-dependent" attenuation (tau attn is not statistically different from tau 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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
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