Department of Neurophysiology, Biomedical Research Center, Osaka University Medical School, Suita 565, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Kimura, Fumitaka, Yo Otsu, and Tadaharu Tsumoto. Presynaptically silent synapses: spontaneously active terminals without stimulus-evoked release demonstrated in cortical autapses. J. Neurophysiol. 77: 2805-2815, 1997. This study addresses the question of whether synapses that are capable of releasing transmitters spontaneously can also release them in an excitation-dependent manner. For this purpose, whole cell patch recordings were performed for a total of 48 excitatory solitary neurons in a microisland culture to observe excitatory autaptic currents elicited by spontaneous transmitter release as well as by somatic excitation. A somatic Na+-spike, induced in response to a short voltage step, evoked excitatory postsynaptic currents (EPSCs) of various amplitudes through the autapses; in some cases, no response was noticeable. To make sure that the recorded autaptic spontaneous EPSCs (sEPSCs) under a voltage clamp resulted from independent release of transmitters and were not associated with action potentials, sEPSCS in the presence and absence of tetrodotoxin (TTX) were compared in six cells. In the presence of TTX the evoked EPSCs were completely eliminated, whereas the sEPSCs were still observed and the amplitude distribution histograms were statistically not different from those recorded in the absence of TTX. A quantitative analysis of the sEPSCs (presumably miniature EPSCs) showed that the amplitude of stimulus-evoked EPSCs did not correlate with either the frequency or median amplitudes of the sEPSCs or the age of the culture. To identify whether the absence of stimulus-evoked response was caused either by conduction failure of excitation along the axons or by impairment of the release machinery that links the terminal depolarization to vesicle exocytosis, we examined whether high K+ and hypertonic solutions could facilitate the spontaneous release of transmitters. Although the hypertonic solution increased the spontaneous release in all cells tested (n = 18), the high K+ solution had a differential effect in increasing spontaneous release, i.e., the cells with larger evoked responses were more readily facilitated by the high K+ solution. Because the high K+ solution induced depolarization of presynaptic terminals, the present results indicated that the smaller evoked responses were due to the larger number of impaired or "silent" presynaptic terminals that were unable to link presynaptic depolarization to transmitter release. In summary, the present experiments provided evidence that at least some of the presynaptic terminals are silent in response to stimuli, while remaining spontaneously active at the same time. Because this phenomenon is due to the lack of sensitivity to depolarization at the terminals, these synaptic terminals seem incapable of linking terminal depolarization to transmitter release.
Synaptic terminals are believed to be capable of releasing transmitters in two distinct ways: action potential-dependent and independent release. The latter form of release is known to produce miniature responses (Fatt and Katz 1952 Culture preparation
Solitary neurons were cultured by using conventional microisland methods (Segal and Furshpan 1990 Electrophysiology
A whole cell patch electrode (5-15 M Analysis of stimulus-evoked EPSC and spontaneous EPSC
The amplitude of stimulus-evoked EPSCs was evaluated by calculation of the mean value between 5 and 15 ms from the peak of the Na+-spike evoked during the short voltage step. For spontaneous EPSCs (sEPSCs), data were searched with a manual peak detection procedure that meets the criteria of peaks with a threshold >3 x SD of the baseline noise level and with a faster rise than the decay phase. The difference between the current from the baseline (2 ms immediately before the onset of each event) and the peak (average of 3 points) was then measured manually. In some experiments events were detected with the use of an event detection program that is a built-in component of the AxoGraph 3.0. Baseline noise was calculated as a difference between the mean of the equivalent baseline periods and an average of three points 1 ms apart from the end of the baseline period. In the experiments that used the high K+ and hypertonic solution, sEPSCs were detected with Fetchan (Axon Instruments), where the first derivative must exceed a threshold of 3 x SD of the background (Malgaroli and Tsien 1992 Analyses of sEPSC amplitude histogram
For some experiments an attempt was made to fit the constructed histograms of sEPSCs to the sum of multiple Gaussian curves. The fit was performed without any constraints except for the number of Gaussian functions (n = 1, 2, 3) that were given for each trial. Each fit was then evaluated with the least-square method and the best results were provided. The entire procedure was performed with the aid of software (Origin, Microcal). The skewness of each histogram was evaluated with the third moment of the distribution (Vautrin and Barker 1994 Staining with neurobiotin
In some experiments recording electrodes contained N-(2-aminoethyl)biotinamide hydrochloride (Neurobiotin; 0.5%) or sulforhodamine (Sigma, 15 µM) to visualize the extent of neurites. After the recording session the cells filled with Neurobiotin were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffered saline (PBS) for 20 min and were then processed with PBS containing 1% Triton-X. Visualization was achieved with the commercially available avidin-biotin-peroxidase method with a combination of diaminobenzidine hydrochloride as the peroxidase substrate (ABC Elite, Vector Laboratories).
Input-output specificity of solitary neurons
The solitary neuron in a culture has a unique circuit; i.e., not only all the inputs for a given solitary neuron arise from the same cell, but also all the outputs terminate again at the same cell. This unique input-output specificity is the result of two important conditions: 1) the existence of only one cell within a restricted area and 2) an overwhelming preference of cell bodies and neurites to stay within the area (Bekkers and Stevens 1991
Stimulus-evoked and spontaneous EPSCs
We took advantage of this characteristic to evaluate the ability to produce EPSCs by somatic excitation in synapses whose existence was confirmed by the appearance of spontaneous activities. A total of 48 excitatory solitary neurons was analyzed electrophysiologically in the present study. A short voltage step applied to the soma through a whole cell pipette induced a Na+-spike, which in turn produced EPSCs. Figure 2A shows an example of a cell that elicited a sizable EPSC after somatic excitation. The EPSC was differentially suppressed by 100 µM APV [an N-methyl-D-aspartate (NMDA) receptor antagonist] and 20 µM CNQX (a non-NMDA receptor antagonist) and was completely eliminated by the simultaneous application of both antagonists, suggesting both NMDA and non-NMDA receptor involvement (Bekkers and Stevens 1991
Partial or complete absence of stimulus-evoked response
The amplitude of evoked responses varied from cell to cell (see Fig. 5 for details). In some cases a somatic excitation failed to produce any noticeable evoked response, although spontaneous events were present and showed similar characteristics to those observed in cells with evoked responses. An example is shown in Fig. 3. An application of antagonists failed to cause any recognizable change after the somatic excitation (Fig. 3A, right), indicating a complete failure of stimulus-evoked responses in this cell. The properties of the spontaneous events, however, such as sensitivity to the antagonists (B), characteristics of the amplitude distribution histogram (C), and the shape of averaged traces (D) were indistinguishable from those of cells with large evoked responses (Fig. 2). The current clamp recording (n = 13) also confirmed the lack of postsynaptic responses after somatic excitation in such cells, where an action potential failed to produce an excitatory postsynaptic potential (data not shown), indicating that the inability to elicit a postsynaptic response was not due to some artificial or instrumental errors.
Effect of TTX on the spontaneous EPSCs
To ensure that autaptic sEPSCs recorded while voltage-clamping presynaptic cell bodies were independent of any form of presynaptic action potentials and thus were identical to what are called miniature EPSCs, it seemed necessary to rule out the possibility that the sEPSCs might have been triggered by local spikes that were not detected by the electrode attached at the soma. For this purpose, sEPSCs were further characterized in the absence and presence of TTX in six cells. As shown in Fig. 4, evoked EPSCs were completely eliminated in the presence of TTX (1 µM), whereas sEPSCs were still observed. The amplitude histograms constructed from sEPSCs in the presence and absence of TTX were not significantly different as determined by Kolmogorov-Smirnov test (P > 0.1) in any of the six cells. This result indicates that autaptic sEPSC during voltage clamp resulted from spontaneous liberation of transmitters and was not associated with presynaptic action potentials.
Lack of correlation between amplitude of stimulus-evoked EPSCs and frequency or amplitude of spontaneous EPSCs
We addressed the question of whether the variability of the amplitude of stimulus-evoked EPSCs could be accounted for by differences either in the number of release sites or in the average amplitude evoked at individual postsynaptic sites. Because it has been suggested that the rate of sEPSCs increases in parallel with the number of terminals stained by an antibody against synapsin I (Gottmann et al. 1994 Sensitivity of spontaneous EPSCs to high K+ solution
We attempted to identify which of the following two possibilities could account for the absence of stimulus-evoked transmission from synapses that are capable of spontaneous release: a conduction block of excitation along the axon or impairment of the release machinery that links terminal depolarization to the transmitter release. An application of high K+ solution is known to enhance transmitter release (Liu and Tsien 1995
The results presented here have demonstrated the existence of synapses that can release transmitters spontaneously, but not in an excitation-dependent way. We have also shown that such silent synapses may result from some impairment of the release machinery especially in linking terminal depolarization to transmitter liberation at presynaptic terminals.
Spontaneous events and evoked responses
The validity of our conclusion depends on the existence of spontaneous events and the evaluation of the amplitudes of stimulus-evoked responses. The median amplitude of spontaneous events was well within the range of the reported values (Bolshakov and Siegelbaum 1995 Conduction block or transmission failure?
Our results could be explained by the deficiencies in either sodium or calcium currents in axons. In other words, we could not completely rule out the possibility that conduction block occurred along axons in cells without stimulus-evoked responses, because in peripheral neurons such as Ia fibers or spinal motoneurons, action potential propagation failure at branching point has been reported (Henneman et al. 1984 Two distinct mechanisms for spontaneous release of transmitters
Our experiments with the high K+ and hypertonic solutions clearly demonstrated the following two points. 1) Spontaneous release may be facilitated through two distinct mechanisms. This is the first experimental evidence showing that synaptic terminals responsive to hypertonic stimulation are not necessarily responsive to depolarizing stimulation. 2) The cells with a smaller amplitude of evoked EPSCs had a lower sensitivity to the high K+ solution. We conclude from these results that the larger amplitude of stimulus-evoked EPSC is accounted for by the larger number of synaptic terminals capable of transmitter release in response to depolarization. We also suggest that the number of synapses are not significantly different among cells with different amplitudes of EPSCs (Fig. 5). Taken together, the number of synapses formed in a given cell might be similar across cells but the number of "active" synapses that release transmitters when depolarized is different from cell to cell, and this variation could underlie the difference in amplitude of EPSCs. Such a possibility that a higher number of silent synaptic contacts might be the basis of differences in synaptic responses has been discussed (Mennerick et al. 1995 What is impaired in the presynaptically silent synapse?
As for the exact location of the impairment, we only know that it should be somewhere downstream between terminal depolarization and vesicle exocytosis. An analogy with studies on synaptogenesis suggests one attractive possibility, namely, the lack of adequate Ca2+ channels in close proximity to releasing sites. During synaptogenesis it is well known that after plating cells in culture there is a time when one can record spontaneous events but not any response in a stimulus-evoked way by activating the neighboring cells (Basarsky et al. 1994 Previously reported presynaptically silent synapses
The existence of silent synapses of presynaptic origin has been suggested in in vitro neuromuscular junctions (Dubinsky and Fischbach 1990 Advantage of single-neuron microculture
Ever since Furshpan et al. (1976)
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). No previous work has ever questioned whether individual synaptic terminals are capable of both types of release except in a limited case (Korn and Faber 1990b
). The simple reason for this is that no adequate preparation was available enabling examination of this important question. This question, however, should be answered before spontaneous events are analyzed to infer quantal changes in stimulus-evoked responses as was done in the studies of long-term potentiation (Malgaroli and Tsien 1992
; Manabe et al. 1992
). In 1976, Furshpan et al. invented a unique culture preparation called microisland, where cell bodies as well as neurites are allowed to grow in a restricted area. Since then this preparation has been used frequently, mainly because it allows a monosynaptic recording to be obtained by using a single electrode with enormous ease (Bekkers and Stevens 1991
; Furshpan et al. 1986
; Mennerick et al. 1995
; Mennerick and Zorumski 1994
; Pan et al. 1993
; Rosenmund et al. 1993
; Segal 1991
; Segal and Furshpan 1990
; Tong and Jahr 1994
). Unlike conventional approaches, experiments with autapses require only a single electrode to identify synaptic pairs whose connections are almost guaranteed. Indeed, this is one of the most important advantages over conventional studies of synaptic transmission, where the first step in experiments is to find a monosynaptically connected pair. This step is an extremely painstaking task even in a slice preparation and, with few exceptions, almost impossible in in vivo preparations. In addition to this advantage, using autapses has a second benefit that is as important as the first. In an autapse, one can record not only a unitary response elicited by a single cell, but also all the events occurring at individual synapses, the total of which supposedly constitutes the unitary response. Therefore if spontaneous synaptic events can be recorded, one could also expect to observe stimulus-evoked responses by excitation of the cell body that is the single source of all synaptic terminals. Thus it could serve as an ideal preparation for addressing the following question: can synapses that are capable of releasing transmitters spontaneously also release them in a stimulus-evoked manner? Taking advantage of autapses formed in a solitary neuron, we report that there are some synapses that can release transmitters spontaneously but not in response to somatic excitation; thus the arrival of an action potential at such a synapse results in a failure of transmitter release. We also found that such silent synapses may be attributable to some impairment of the release machinery that links presynaptic depolarization to transmitter release at the presynaptic terminals.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). A piece of visual cortex was removed from neonatal rats (typically <3-days old), enzymatically dissociated with papain (20 U/ml), and triturated with a fire-polished glass pipette. Neurons were plated on previously prepared glial islands grown on collagen dots placed on an agarose sheet. Cells were grown in a solution based on Eagle's Minimum Essential Medium supplemented with 5% rat serum (Baughman et al. 1991
; Otsu et al. 1995
). Most of the cultures were raised in the presence of an ionotropic glutamate receptor antagonist kynurenate (0.1-1 mM) to inhibit glutamate-mediated excitotoxicity (Choi et al. 1987
; Furshpan and Potter 1989
; Obrietan and Van Den Pol 1995
). Recordings were made at 9-147 days after plating.
resistance) was used to record synaptic currents in a voltage-clamp mode with either an Axoclamp-2B (continuous voltage clamp) or an Axopatch 200A without series resistance compensation. When the Axoclamp-2B was used an additional test for the presence of an evoked excitatory postsynaptic potential (EPSP) after an action potential was routinely performed in the current clamp mode. The cells were clamped at
70 mV for recording EPSCs unless otherwise noted. A short voltage step from holding potential to
10 to +40 mV for 2-3 ms was applied to elicit a somatic Na+-spike. The perfusing solution, unless otherwise noted, contained the following (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 10 glucose, 10 µM glycine, and 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES; pH 7.4), but no addition of Mg2+. For part of the experiments, the same solution with 1 mM MgCl2 added was used (see RESULTS). Osmolality was adjusted to between 325 to 335 mosm by adding sucrose when necessary. This rather high osmolality was preferred to match not only that of our culture medium (320-330 mosm) but also that of the experiments with the high K+ solution. The high K+ solution contained 30 mM instead of the 4 mM KCl used for the normal solution, which usually had an osmolality of ~330 mosm. In one case it was >335 mosm, so that the entire solution was diluted by a factor of 0.94. Drugs were applied with a local perfusion system equipped with a Y-shaped tube. We used three different kinds of electrode solutions to make sure that the absence of evoked responses was not due to some artifact. The solutions mainly used were as follows (in mM): 150 K-methane sulfonate (or 130 Cs-methane sulfonate), 10 HEPES, 10 KCl (or 10 CsCl), 0.5 EGTA, 5 MgATP, and 1 Na2GTP. To eliminate the possibility that the principal anion had some undesirable effect that might prevent the elicitation of EPSCs, K-gluconate (130 mM) was also used without changing the concentration of the rest of the ingredients. All solutions were adjusted to pH 7.3 and 320 mosm. Because no noticeable differences were seen in the ability to produce evoked responses among these three electrode solutions, all the data were combined. The signal was digitized at 10 kHz, filtered at 1-3 kHz, and analyzed with a pClamp 6 or an AxoGraph (Axon Instruments) by using an IBM-PC clone computer or Macintosh and stored on DAT tape (PC-108M, Sony Magnescale) for further analysis. The culture medium was obtained from GIBCO, papain from Worthington, D,L-2-amino-5-phosphonovaleric acid (APV), kynurenate from Sigma, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Tocris.
) to be identified as a candidate event. The detection was then visually inspected to exclude false events caused by an artificial source such as environmental noise.
; Vautrin et al. 1993
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
; Furshpan et al. 1976
; Segal 1991
). An example of such a neuron is demonstrated in Fig. 1, where the cell was injected with neurobiotin to visualize its processes. We confirmed that neuronlike processes were evident only within the microculture and that no neuronal processes were identified outside the microcultures in any of the dye-injected neurons by either Neurobiotin (n = 6) or sulforhodamine (n = 2). These findings conform with previous studies (Bekkers and Stevens 1991
; Furshpan et al. 1976
; Segal 1991
).
View larger version (112K):
[in a new window]
FIG. 1.
Photomicrographs of an example of a solitary neuron. A: phase contrast picture; only a single cell was identified in this circular area. B: bright field picture of the same field as in A; growth of neurites as well as cell body is restricted in this area. Cell was injected with Neurobiotin. Calibration bar = 50 µm; 26 days in culture.
; Segal 1991
). Spontaneous events in the same cell are shown in Fig. 2B. The following four points support that these events resulted from the spontaneous liberation of excitatory transmitters from presynaptic terminals. 1) The spontaneous events were not associated with somatic Na+-spikes that would have appeared in close temporal association if these events were driven by Na+-spikes (note that the recording was made from the "presynaptic" cell body). 2) These events were also suppressed by APV and CNQX at the same concentration as the evoked EPSCs (Fig. 2B1, right column), suggesting that they were mediated by glutamate as a transmitter. 3) An amplitude distribution histogram constructed from these spontaneous events (Fig. 2C) exhibited the typical characteristic of miniature EPSC distribution (Bekkers et al. 1990
; Bekkers and Stevens 1989
), i.e., a single peak skewed to the larger events with a mean amplitude of 6.9 ± 3.1 pA (mean ± SD, n = 465). Consistent with previous reports (Korn and Faber 1990b
; Ulrich and Luscher 1993
), the histogram seemed to consist of the sum of two Gaussian distributions. 4) The average of 24 such spontaneous events showed the typical shape of EPSCs resulting from non-NMDA and NMDA receptor mediation (Fig. 2D). The average consisted of a fast rise and a fast decay followed by a slow decay; the decay phase was fitted to a double exponential curve and the time constants were 2.6 and 24.4 ms, consistent with the range reported for EPSCs mediated by non-NMDA and NMDA receptors in the visual cortical neurons (Hestrin 1992
, 1993
; Stern et al. 1992
).
View larger version (28K):
[in a new window]
FIG. 2.
Example of cells with evoked response after somatic excitation. A: autaptic excitatory postsynaptic currents (EPSCs) suppressed by D,L-2-amino-5-phosphonovaleric acid (APV, 100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) differentially. Averages of 4 responses have been superimposed. B1 (right column): spontaneous autaptic EPSCs were also suppressed by the simultaneous application of APV and CNQX. B1 (left column): , events selected for further analysis; *, expanded as shown in B2. B2: illustration of how each event in the expanded period was measured. C: amplitude distribution histogram of spontaneous events exhibiting the typical distribution of miniature responses, i.e., a single peak skewed to larger events (skewness = 5.9). Distribution was best fitted to the sum of two Gaussians with peaks at 5.8 and 8.7 pA. Inset: noise distribution. D: average of 24 spontaneous EPSCs (sEPSCs) that had 2 decay phases. Time constants obtained from a double exponential fit were 2.6 and 24.4 ms; 28 days in culture.
View larger version (17K):
[in a new window]
FIG. 5.
Relationship between the amplitude of evoked response and various aspect of sEPSCs. In each graph plotted against the amplitude of evoked EPSC (abscissa) are mean frequency (A), median amplitude (B), and age of the culture (C).
View larger version (21K):
[in a new window]
FIG. 3.
Example of cells without evoked response after somatic excitation. A: short voltage step applied to cell body failed to elicit any detectable response. B: a number of spontaneous events were recorded in the normal solution, which were suppressed in the presence of APV and CNQX (right column). , events selected for further analysis. C: amplitude distribution histogram of the spontaneous events showing distribution of the typical miniature. Mean amplitude was 5.6 ± 1.4 (SD, n = 424); skewness was 2.0. Distribution was best fitted to the sum of 2 Gaussians with peaks at 5.0 and 6.8 pA. Inset: noise distribution. D: average of randomly collected 17 EPSCs was very similar to that from the cell with evoked response (Fig. 2D). Time constants for the decay phase were 2.3 and 17.0 ms; 40 days in culture.
View larger version (22K):
[in a new window]
FIG. 4.
Distribution of spontaneous autaptic EPSCs in the absence of tetrodotoxin (TTX) is not statistically different from that in the presense of TTX. Stimulus-evoked response (left column), sEPSCs (middle), and amplitude histograms with cumulative plots (right) recorded from a solitary neuron in the absence (top row) and presence (bottom row) of TTX (1 µM); 70 days in culture.
), the frequency of spontaneous release should roughly indicate the total number of synaptic sites on a given neuron. Therefore the relationship of the amplitude of the evoked response to both the frequency as well as the median amplitude of spontaneous events was analyzed in 26 cells. The result is shown in Fig. 5, A and B. There was no significant linear correlation between the frequency (A) or median amplitude (B) of spontaneous events and the amplitude of stimulus-evoked responses after somatic excitation (r = 0.08 and 0.06, respectively; P > 0.1). We examined whether the appearance of stimulus-evoked responses depends on the age of the culture, but as shown in Fig. 5C, again no significant linear correlation was found between the age and the amplitude of evoked responses (r = 0.17, P > 0.1). These results indicate that properties of a given neuron such as the number of synaptic sites, the average amplitude of sEPSC, or the culture age do not necessarily explain the variability of its ability to produce stimulus-evoked responses.
; Malgaroli and Tsien 1992
) by inducing presynaptic depolarization, which in turn triggers successive steps leading to vesicle exocytosis. Thus if a conduction block occurs at the cells without evoked responses, the high K+ solution should be able to enhance the release, whereas if impairment of the release machinery underlies the failure of evoked release, the high K+ solution should fail to facilitate the release. In the latter case, however, the possibility remains that the failure of facilitation results from the lack of readily releasable presynaptic terminals. This possibility can be verified by the application of a hypertonic solution that is thought to induce a release of transmitters in a depolarization independent manner (Fatt and Katz 1952
).
View larger version (31K):
[in a new window]
FIG. 6.
Spontaneous EPSCs of cells with evoked responses are sensitive to the high K+ as well as to the hypertonic solution. A: superimposed traces of EPSCs evoked by somatic excitation. B-D: recordings obtained with control (B), hypertonic (C), and high K+ solution (D); 19 days in culture. , identified events. Note: in C and D events marked only on top traces.
View larger version (32K):
[in a new window]
FIG. 7.
Spontaneous EPSCs of cells without evoked responses are sensitive to the hypertonic solution but insensitive to the high K+ solution. A: superimposed traces after somatic excitation. B-D: recordings obtained with control (B), hypertonic (C), and high K+ solution (D). This cell was a sister culture of the cell shown in Fig. 6; 18 days in culture. , identified events. Note: in C events marked only on the top trace.
View larger version (12K):
[in a new window]
FIG. 8.
Relationship between the sensitivity of sEPSCs to high K+ solution and amplitude of evoked response. Ratios of sEPSC frequency with the high K+ solution to that with the normal solution are plotted against the amplitude of evoked response. Frequency of sEPSCs of the cells with smaller evoked responses were less sensitive to stimulation with the high K+ solution.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Chavez-Noriega and Stevens 1994
; Malgaroli and Tsien 1992
). Although the blockade of the spontaneous events by APV and CNQX indicated that they were induced by the activation of glutamate receptors, it still seems important to consider the possibility that glutamate was not released from synaptic terminals in close opposition to the postsynaptic membrane, but from some other sources such as glial cells or even metabolic pools from the neighboring dead cells. This conclusion seems less likely, however, because the averaged waveforms of spontaneous events showed the typical shape of EPSCs with reasonable decay time constants, which would not result from the binding of receptors with free glutamate in the medium.
; Tong and Jahr 1994
). Thus an EPSC peak, if present, would be detectable. Second, we evaluated the amplitude of EPSCs not by the peak amplitude but by calculating the average value of EPSCs between 5-15 ms from the peak of the Na+-spike. Therefore if any EPSCs were induced, the value would not be zero. Third, current clamp recordings confirmed the lack of stimulus-evoked responses in the cells that had been judged as silent in the voltage-clamp mode. Finally, the lack of change in the recordings after stimuli during the application of the antagonist against glutamate receptors additionally supports the absence of stimulus-evoked responses as shown inFig. 3A.
; Parnas 1972
; Smith 1980
). One direct way of ruling out this possibility would be to record from the terminals and postsynaptic membrane extracellularly to locate a site where presynaptic activity does not elicit a postsynaptic response. Alternatively, a strong support for our result would be if a focal application of high K+ solution (Liu and Tsien 1995
) to individual synaptic boutons could locate a site where no response could be induced.
; Pun et al. 1986
). The present results seem to support this possibility.
; Gottmann et al. 1994
; Kraszewski and Grantyn 1992
; Legendre et al. 1988
). Also worth mentioning, the application of TTX, which reduces the rate of spontaneous events in mature synapses, had no effect on the cells within 10 days after plating, suggesting that until then none of the spontaneous release was driven by the existing presynaptic sodium spikes (Dubinsky 1989
). Basarsky et al. (1994)
indicated that the appearance of a stimulus-evoked response temporally almost coincides with the alteration of the expression of Ca2+ channels from L-type to N-type. Several lines of evidence have demonstrated that presynaptic Ca2+ channels change during development into the one responsible for transmitter release (Basarsky et al. 1994
; Scholz and Miller 1995
; Verderio et al. 1995
).
; Hartzell 1988
; Reuter 1983
; Trautwein and Hescheler 1990
; Tsien 1983
). Interestingly, the involvement of cAMP was also implicated in activating presynaptically silent synapses (Dubinsky and Fischbach 1990
) and in increasing transmitter release (Chavez-Noriega and Stevens 1994
).
; Wojtowicz et al. 1991
) as well as in the central nervous system, both in vivo (Charpier et al. 1995
; Faber et al. 1991
; Korn and Faber 1990a
) and in vitro (Pun et al. 1986
). A few of these studies provided evidence suggesting that only 20-25% of the ultrastructurally identified synapses release transmitters in a stimulus-evoked way (Charpier et al. 1995
; Lin and Faber 1988
; Pun et al. 1986
). An interesting question is whether those previously reported silent synapses were able to release transmitters spontaneously. Although those studies could not possibly answer this question because of the lack of adequate preparation, the following two findings suggest the possibility that these synapses could also release transmitters spontaneously. First, extensive electron microscopic studies have revealed no obvious distinction between such silent synapses and "normal" synapses (Furshpan et al. 1986
; Pun et al. 1986
; Sur et al. 1994
). Second, the silent synapses can be activated quite readily by high-frequency stimulation (Charpier et al. 1995
; Lin and Faber 1988
; Wojtowicz et al. 1991
) or a treatment with cAMP activator (Dubinsky and Fischbach 1990
). In addition, Charpier et al. (1995)
reported that the number of synapses found in a silent cell is not significantly different from that in a "potent" cell that always generates stimulus-evoked responses; this finding is in good agreement with our results showing no obvious correlation between the amplitude of evoked responses and the frequency of spontaneous events (Fig. 5).
invented a single-neuron microculture, this preparation has been used repeatedly for a better understanding of the several aspects of synaptic transmission (Bekkers and Stevens 1991
; Furshpan et al. 1986
; Mennerick et al. 1995
; Mennerick and Zorumski 1994
; Pan et al. 1993
; Rosenmund et al. 1993
; Segal 1991
; Tong and Jahr 1994
). Accumulated evidence from these previous works suggests that autaptic transmission is indistinguishable from normal synaptic transmission. For example, Segal (1991)
showed that both inhibitory and excitatory solitary neurons are capable of forming autapses, where transmission is mediated by the GABAA receptor and by a combination of NMDA and non-NMDA receptors, respectively. Bekkers and Stevens (1991)
further demonstrated the following aspects: 1) both excitatory transmission (with NMDA and non-NMDA components) and inhibitory transmission are evident with similar permeation, kinetic, and pharmacological properties as those of normal synaptic transmission; 2) miniature EPSCs (mEPSCs) are observed with a mean size and amplitude distribution similar to those of conventional synaptic excitatory miniature EPSCs; and 3) transmission through excitatory autapses seems quantal. The participation of both NMDA and non-NMDA receptors in the excitatory autapse was confirmed repeatedly afterwards (Mennerick et al. 1995
; Rosenmund et al. 1993
; Tong and Jahr 1994
). Further details of mEPSCs such as amplitude, rise time, decay time constant, and frequency were recently reported to be quite similar to those of normal synaptic transmission (Mennerick et al. 1995
). In addition, as we have demonstrated here, the unique input-output specificity associated with the solitary neuron allows the study of the important relationship between unitary postsynaptic response and mEPSCs. Taking all these considerations into account, the autaptic preparation serves as an excellent device to study important properties of synaptic transmission.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors are especially grateful to Drs. Y. Yoshimura, M. Yamashita, and Y. Oda for helpful discussion and comments throughout the experiments and also to Drs. J. Vautrin and J. L. Barker for valuable discussion for part of this experiment. We also thank L. Voronin for critical reading of the earlier version of this manuscript and Y. Frégnac for providing the protocol for neurobiotin staining.
This work was supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture to T. Tsumoto and by Narishige Neuroscience Research Foundation to F. Kimura.
![]() |
FOOTNOTES |
---|
Present address of Y. Otsu: Dept. of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951, Japan.
Address for reprint requests: F. Kimura, Dept. of Neurophysiology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita 565 Japan. E-mail: fkimura{at}nphys.med.osaka-u.ac.jp
Received 13 May 1996; accepted in final form 2 January 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|