Changes in Quantal Size Distributions Upon Experimental Variations in the Probability of Release at Striatal Inhibitory Synapses

Jan C. Behrends and Gerrit ten Bruggencate

Department of Physiology, Universität München, 80336 Munich, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Behrends, Jan C. and Gerrit ten Bruggencate. Changes in quantal size distributions upon experimental variations in the probability of release at striatal inhibitory synapses. J. Neurophysiol. 79: 2999-3011, 1998. Postsynaptic inhibitory gamma -aminobutyric acid-A (GABAA)-receptor-mediated current responses were measured using simultaneous pre- and postsynaptic whole cell recordings in primary cell cultures of rat striatum. Substitution of Sr2+ for extracellular Ca2+ strongly desynchronized the inhibitory postsynaptic currents (IPSCs), resulting in a succession of asynchronous IPSCs (asIPSCs). The rise times and decay time constants of individual evoked asIPSCs were not significantly different from those of miniature IPSCs that are the result of spontaneous vesicular release of GABA. Thus asIPSCs reflect quantal transmission at the individual contacts made by one presynaptic neuron on the recorded postsynaptic cell. Increasing the concentration of Sr2+ from 2 to 10 mM and decreasing that of Mg2+ from 5 to 1 mM produced an increase in the frequency of asIPSCs consistent with an enhancement of the mean probability of release (Pr). At the same time the amplitude distribution of asIPSCs was shifted toward larger values, whereas responses to exogenously applied GABA on average were slightly decreased in amplitude. Application of the GABAB-receptor agonist baclofen (3-10 µM) strongly reduced the frequency of asIPSC, consistent with a decrease in Pr, and led to a shift of the amplitude distribution toward smaller values. Baclofen had no effect on responses to exogenously applied GABA. In summary, our data suggest that at striatal inhibitory connections the weight of single contacts may be controlled presynaptically by variation in the amount of transmitter released.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Synaptic efficacy in the CNS is known to be modulated through a variety of mechanisms that affect the coupling between a presynaptic impulse and transmitter release (i.e., the probability of release Pr). This presynaptic modulation occurs at the individual terminals established by a presynaptic axon on the postsynaptic cell. For instance, many drugs or transmitters have been shown to enhance or depress Ca2+-dependent transmitter release via regulation of the activity of voltage-dependent Ca2+-channels (for review see Verhage et al. 1994). In principle, such mechanisms may provide a means to rapidly adjust the gain of an individual synaptic contact by, for instance, the local activation of presynaptic receptors.

However, statistical analyses of fluctuations in the amplitude of postsynaptic responses have suggested a general model where single sites operate in an all-or-nothing fashion (Redman 1990), so that locally graded presynaptic regulation of transmission would not be possible. In this view, either one synaptic vesicle at most is released per impulse and emptying of its transmitter content is always complete (1-vesicle hypothesis) (Bekkers et al. 1990; Bekkers and Stevens 1995; Frerking et al. 1995; Stevens and Wang 1995) or postsynaptic receptors are always saturated by the smallest amount of transmitter that can be released (saturation hypothesis) (Edwards 1995; Edwards et al. 1990; Jonas et al. 1993; Kullmann 1993). According to both views, presynaptic modulation exclusively affects reliability (i.e., the probability of a contact site's taking part in transmission at all); the weight of a single contact (the postsynaptic response generated in case it is active) will not change with Pr. One simple consequence of all-or-nothing transmission for information transfer is that finely graded regulation of synaptic strength would require many contacts in a connection. However, the large number of vesicles in a presynaptic terminal may, in principle, provide it with sufficient flexibility ofits own.

To test the all-or-nothing hypothesis, it is necessary to record responses to release events from single sites under conditions that allow Pr to be readily modified and measured. We have recorded asynchronous inhibitory postsynaptic currents (asIPSCs) evoked in the presence of extracellular Sr2+ and determined changes in their frequency and their amplitude distributions on modifying conditions that determine Pr. Our present report demonstrates that asIPSCs represent responses to gamma -aminobutyric acid (GABA) release from single contacts and that experimental changes in Pr affect not only the frequency but also the amplitude distribution of these events. We conclude that the synapses studied here are not likely to function in an all-or-nothing fashion.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell culture and electrophysiology

Low-density dissociated cell cultures of striatal neurons were prepared exclusively from the lateral ganglionic eminence dissected from rat embryos at gestational day 16-18. Culture methods given in Gottmann et al. (1994) were followed. Cultures were used for recordings from 10 to 25 days in vitro, when the large majority of cells stained positive for GABA (not illustrated). Paired recordings of synaptically coupled neurons were obtained under direct visual control on a Zeiss IM35 inverted microscope using borosilicate pipettes (1.5-2 µm OD, open tip resistance 3-5 MOmega ) filled with a solution of the following composition (in mM): 110 KCl, 5 MgCl2, 0.6 ethylene glycol-bis(beta -aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 2 Na-adenosine 5'triphosphate (Na-ATP), pH 7.3 with 230 mOsm KOH. Control extracellular solution contained (in mM) 125 NaCl, 1 KCl, 1 MgCl2, 20 HEPES, and 2 CaCl2, pH 7.35 with 270 mOsm NaOH. All recordings were done at room teperature (22-25°C). IPSCs were evoked by short (3-5 ms) depolarizing step commands to the presynaptic neuron in voltage clamp mode (holding potential near -70 mV) that elicited action currents. Stimuli were delivered every 5-10 s. Solutions surrounding the recorded cells were exchanged using local application through a Y-tube. When recording miniature IPSCs (mIPSCs), their frequency was sometimes enhanced by local application of an extracellular solution in which 14 mM of NaCl was replaced with KCl and to which 1 µM of tetrodotoxin (TTX; Sigma, Munich, Germany) was added to block action potentials. Two EPC-7 patch clamp amplifiers (List Medical, Darmstadt, Germany) were used for recording. Series resistance (<20 MOmega ) was monitored at regular intervals and not compensated for. Output signals were filtered at 3 kHz and digitized on-line at 24 kHz with a NB-MIO16L 14-bit AD-converter (National Instruments, Austin, TX) in a Macintosh 8100/100 computer.


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FIG. 1. Desynchronization by Sr2+ substitution for Ca2+ of postsynaptic GABAergic responses. A: presynaptic action current (a) and superimposed inhibitory postsynaptic currents (IPSCs; b, n = 10) recorded in the presence of 2 mM Ca2+ in the extracellular solution. Each trace is 630 ms long. Inset: onset of IPSCs shown on expanded timescale. c: superimposed traces (n = 10) recorded in the absence of presynaptic stimulation. Ba-Bc: as in A, but after replacement of Sr2+ for Ca2+. Note the dissociation of the responses into a succession of individual asynchronous IPSCs which resemble miniature IPSCs. The 100-ms calibration bar applies to Aa-Ac and Ba-Bc; the 5-ms calibration bar to the insets in Ab and Bb. Amplitude calibrations are the same for Aa and Ba (1 nA) and Ab, Ac, Bb, and Bc (200 pA), respectively. C: superimposed averages of IPSCs shown in Ab (Ca2+) and Bb (Sr2+), respectively; the latter is also shown scaled to the peak of the Ca2+ response (Sr2+ scaled) to facilitate kinetic comparison. Thick lines, double-exponential fits. D: superimposed time integrals of averaged responses in C. Each mean time course is also shown ±SD. Note that in Sr2+-containing solution, the total synaptic charge builds up more slowly but exceeds the control value after ~400 ms.

Event detection and analysis

asIPSCs were automatically detected on the basis of a slope criterion, amplitudes and times to peaks were measured, and individual decays were fitted with single time constants after digital Bessel filtering at 2 or 1 kHz employing DETECTiVENT software (Ankri et al. 1994) written in LabView (National Instruments). Operator interference during detection was limited to the setting of detection parameters and filter frequency, which were kept constant for a given experiment. Amplitudes of asIPSCs occurring on decays of preceding events were corrected using the modal value of the distribution of decay time constants (Ankri et al. 1994). Poststimulus time histograms were calculated for all experiments to ascertain that events were correlated with the stimulus. The reliability of detection was ascertained by superimposition of the data points for onset and peak of individual events on the original recording traces. This process often resulted in the identification of early periods (20-300 ms after stimulus) where automatic detection of single asIPSCs was unreliable because of high event frequency; these early periods were then excluded from further analysis.

For superimposition and averaging of asIPSCs (except in Fig. 2B), the data trace before and after each event was reconstructed by using the measured parameters (time of onset, time to peak, amplitude, and decay time constant) of all other asIPSCs occurring in the same trial. This reconstructed trace was subtracted from the original trace, resulting in the isolation of the event of interest. This procedure allowed the visual comparison of amplitudes and waveforms of individual and averaged events that would otherwise be distorted by preceding or following signals.


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FIG. 2. Detection and characteristics of asIPSCs. A: sample traces (1,260-ms long) of desynchronized postsynaptic responses (2 mM Sr2+). Circles mark onsets and peaks of automatically detected individual asIPSCs. B: superimposed single asIPSCs from the experiment shown in A, aligned on their rising phases. C: relative frequency histogram (bars, left ordinate; bin size 1.5 pA) and estimated cumulative probability distribution (thick line, right ordinate) of asIPSC amplitudes (394 events). Both graphs were fitted with a Gaussian raised to the 3rd power and a gamma distribution (thin lines). There is no visible difference between the 2 fits. D: relationship between times to peak and amplitudes. Top panel: events from 20 ms after stimulus. Bottom panel: events from 200 ms. Note that there is no difference in the correlation (correlation coefficients are 0.73 and 0.71, respectively).

Data were further analyzed and figures prepared with IGORPro (Wavemetrics, Lake Oswego, OR) software. Statistical treatment was performed with StatView software (Abacus Concepts, Berkeley, CA). Average values are given as mean ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Characteristics of asynchronous evoked IPSCs

Figure 1 shows results of a typical experiment in which, during a paired pre- and postsynaptic recording, the normal extracellular solution was exchanged against one containing Sr2+ instead of Ca2+. Figure 1A shows a presynaptic action current elicited by a short depolarizing voltage command (a) and several superimposed postsynaptic current responses (b). These were sensitive to the GABAA-receptor antagonist bicuculline whenever tested (data not shown) and thus represent GABAergic inhibitory Cl- currents (IPSCs). After equimolar (2 mM) replacement of Ca2+ with Sr2+ (Fig. 1B), the peak amplitudes of the IPSCs (b) were reduced markedly and a late component became apparent that often lasted for >1 s and consisted of individually discernible, small, and asynchronous current events in a succession. As shown in the expanded view (cf. Fig. 1, Ab and Bb, insets) the early component was desynchronized as well, with the times to peak of the postsynaptic responses fluctuating to a much larger extent than under control conditions. Traces recorded in the absence of presynaptic stimulation (n = 10) show the low frequency of spontaneous events (Fig. 1, Ac and Bc) that, therefore, did not significantly contaminate the stimulus-induced response.

Thus fast inhibitory GABAergic transmission appears desynchronized after substitution of Sr2+ for Ca2+, much like transmission at the neuromuscular junction (Bain and Quastel 1992; Dodge et al. 1969; Meiri and Rahamimoff 1971; Zengel and Magleby 1980) and at glutamatergic hippocampal synapses (Abdhul-Ghani et al. 1996; Goda and Stevens 1994; Oliet et al. 1996).

Comparison of the response averages in Fig. 1C reveals a strong enhancement of the slower phase in the double-exponential decay of the IPSC in Sr2+- with respect to Ca2+-containing extracellular solution. Finally, Fig. 1D illustrates the time course of mean synaptic charge transfer during the responses. This analysis shows that transmission was slower in Sr2+ with respect to control but total charge transfer began to exceed control values after ~400 ms, indicating that transmitter release activated by Sr2+ is not less intense but spread out in time with respect to that induced by Ca2+ influx. Thus the individual components of the asynchronous response that are particularly visible in its later phase are likely to represent dissociated, evoked transmission at individual contact sites.


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FIG. 3. Evoked asIPSCs are kinetically indistinguishable from mIPSCs. Aa-Ac: desynchronized responses recorded in presence of 2 mM SrCl2. Ad-Af: mIPSCs recorded in 1 µM tetrodotoxin, 15 mM KCl, and 2 mM CaCl2 (see METHODS). Trace length is 1,260 ms. Ba: superimposed single asIPSCs. Bb: superimposed mIPSCs (n > 50). C: frequency histograms for times to peak of asIPSCs (top panel, n = 351) and mIPSCs (bottom panel, n = 267). Bin, 2 pA. D: frequency histogram for amplitudes of asIPSCs (top panel) and mIPSCs (bottom panel). Bin, 0.1 ms. All data shown are taken from the same experiment.

Figure 2A illustrates the automatic detection (see METHODS) of the individual asIPSCs in the Sr2+-dependent response from another recording. The individually detected asIPSCs are shown superimposed in Fig. 2B on a fast timescale. This superimposition is quite suggestive of equidistant preferred amplitudes, and some peakedness is apparent in the corresponding amplitude histogram shown in Fig. 2C. However, amplitude histograms with equidistant peaks did occur in this study only in a minority (~20%) of cases. In fact, as shown here (Fig. 2C), they were well fitted with continuous distributions such as third-power Gaussians (Bekkers et al. 1990) or gamma distributions (McLachlan 1978). The goodness of fit with power Gaussians was largely insensitive toward the power used, provided it was >2.

The observed relationship between times to peak and amplitudes (Fig. 2D, top panel) is the reverse of what would be expected if cable filtering contributed dominantly to the variation of amplitudes, but might result if larger events were due to undetected superposition of multiple smaller events. However, because the same relationship is obtained when the analysis is confined to the late, low-frequency component of the response where such collisions are unlikely (Fig. 2D, bottom panel), this is clearly not the case. Such positive relationships were obtained experimentally (Paulsen and Heggelund 1996; Ropert et al. 1990) as well as from simulated events with homogeneous times to peak (Ankri et al. 1994) and might therefore be due to the detection method used.

If asIPSCs, like mIPSCs, are indeed caused by single release events, they would permit study of evoked transmission events at single release sites and testing for effects of changes in Pr on their amplitude distributions. Direct comparison of asIPSCs and spontaneous mIPSCs recorded in the presence of 1 µM TTX and 15 mM KCl (see METHODS) from the same neuron (n = 3) showed a close similarity in their time courses. An example is shown in Fig. 3, A-C. Overall, rise times (0-100%) averaged 2.23 ± 0.20 (SE) ms for asIPSCs recorded in the presence of 2 mM Sr2+ and 1 mM Mg2+ (n = 13 pairs) and 2.20 ± 0.42 ms for mIPSCs (n = 6 cells). The decays of both asIPSCs and mIPSCs could be fitted with single time constants of 22.37 ± 2.61 ms and 19.51 ± 3.72 ms, respectively. These results allow the conclusion that asIPSCs, like mIPSCs, are responses to single events of transmitter release at individual contact sites. The shape of amplitude distributions of asIPSCs was similar to that of mIPSCs (Fig. 3D); at holding potentials between -70 and -80 mV, amplitudes of asIPSCs ranged from5.65 ± 0.75 pA and 268.97 ± 59.20 pA with a mean of44.40 ± 4.90 pA and distributions were positively skewed with coefficients of variation ranging from 50.3 to 130.1%(mean = 78.5%). mIPSCs had skewed amplitude distributions between 5.70 ± 0.96 pA to 140.47 ± 40.85 pA with a mean of 29.83 ± 3.88 pA and coefficients of variation between 59.2 and 93.3% (mean = 68.9%).

The experiments reported thus far have established that evoked asIPSCs at a single monosynaptic connection are kinetically indistinguishable from quantal mIPSCs. Furthermore, the above comparison of the coefficients of variation in amplitude indicates that quantal sizes vary at least as much at single connections (asIPSCs) as in a population of contacts originating from multiple unidentified presynaptic neurons (mIPSCs).

Changes in Pr affect asIPSC-amplitude distributions

The purpose of this study was to test a prediction common to both variants of the all-or-nothing hypothesis, namely that quantal size distributions are insensitive to changes in release activity at presynaptic terminals. To this end, Edwards (1995) has suggested recording miniature events under different conditions of Pr. However, in CNS neurons one cannot be assured that miniatures are sampled from the same population of contacts under all conditions and that all contacts are sensitive to the manipulations used to vary Pr. This problem is certainly reduced in the case studied here, where all asIPSCs originate from contacts made by the same presynaptic cell. Furthermore, as indicated by the comparison of the total charge transfer in Fig. 1D, asIPSCs (in contrast to mIPSCs) are due to vigorous activation of the release process; i.e., the total quantal content of the response is similar to normal evoked transmission.

Altering the concentration ratio of the extracellular divalent cations Sr2+ and Mg2+ is a convenient way to induce rapid changes in Pr (Abdul-Ghani et al. 1996; Goda and Stevens 1994), which can be assessed as changes in the frequency of asIPSCs occurring after a stimulus. Amplitudes of individual events may then be measured to determine whether quantal size remains stable.

In principle, however, any increase in frequency may produce an artifactual shift in the amplitude distribution. First, two or more individual asIPSCs occurring at intervals shorter than the discriminative threshold of the detection will be falsely detected as one with a correspondingly higher amplitude. Second, small events riding on the decay of larger ones may be missed because they fail to reach the slope threshold (Ankri et al. 1994). The propensity for collisions as well as selective loss of small events is frequency dependent and will therefore decrease throughout an asynchronous response as the frequency of events diminishes. Furthermore, the presence of a significant fraction of "multiples" caused by collisions is expected to be signaled by an excess of higher values in the distribution of times to peak (Ankri et al. 1994). Thus, to avoid these sources of systematic error, we 1) excluded from analysis early, high-frequency periods of the responses, where it was obvious on visual inspection that some closely spaced events were not discriminated or that small events were missed (see METHODS) and 2) discarded experiments where, despite elimination of obviously unreliable data sections, the distribution of times to peak was significantly affected [Kolmogorov-Smirnov (KS) test] after a change in Pr. Because of its crucial importance for the reliability of our amplitude measurements, validation of this procedure is documented in Figs. 4 and 5. Traces of data closely resembling those recorded (see legends for details) were generated by computer with asIPSCs occurring at a mean frequency of 6 Hz (Fig. 4A) or 60 Hz (Fig. 4B). By using the procedure outlined above, we obtained significant differences between amplitude distributions only if the simulated distribution had indeed been different (Fig. 6C). Therefore exclusion of high-frequency portions of data and matching of time to peak distributions appears effective in supressing the systematic error associated with superpositions of events. This is due to a tight correspondence between artifactual, frequency-dependent shifts in the distributions of amplitudes and of times to peak and, in turn, between these shifts and an independent estimate of event discrimination: Fig. 5A shows latency histograms obtained from the data shown in Fig. 4, A and B. Comparison with the simulated time course of the frequency (smooth lines) shows that events are lost during the first 200 ms in the case of the 60-Hz data; no such deviation is present at 6 Hz. Figure 5, B and C, shows the effect of progressive elimination of early events on the amplitude and time to peak distributions, respectively. To begin with, both distributions are significantly different (KS test: P < 0.05) from the low-frequency control. As expected from Fig. 5A, elimination of the first 200 ms, but not of the first 100 ms, of data are sufficient to remove the significant differences for both measures (P > 0.1). Thus changes in the distribution of times to peak are closely linked to and can be used as an indicator for frequency-dependent artifactual changes in amplitude distributions. Figure 5D corroborates this conclusion by showing a perfect correlation beween the test parameter Dmax of the KS-test comparison for times to peaks and amplitudes. In summary, these analyses of simulated data have shown that the precautions taken are sufficient to reduce the effects of frequency-dependent detection error to below the level of significance.


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FIG. 4. Validation of detection procedure using simulated data. Asynchronous synaptic responses were generated at 6 Hz (A) and 60 Hz (B) with a digitization rate of 24 kHz. Time to peak of asIPSCs was 2.2 ms; decay time constant was 20 ms. Their probability of occurrence decreased with time constant of 200 ms. The amplitude distribution was a binomial with q = 10 ± 3 (SD) pA and n = 15 and could be changed by varying the binomial parameter p (pb). Noise was added with an SD of ±2 pA. C: comparison of cumulative amplitude distributions obtained under different conditions using the procedures outlined in the text to suppress systematic error resulting from superposition of high-frequency events. Note that the 2 distributions with pb = 0.05 were not significantly different (Kolmogorov-Smirnov (KS) test: P > 0.1]. All other comparisons resulted in highly significant differences (KS test: P < 0.0001).


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FIG. 5. Significant changes in the distribution of times to peak accompany artifactual shifts of amplitude distributions with increases in frequency. A: latency histogram of simulated asIPSCs at 6 Hz (closed bars) and 60 Hz (open outline). Smooth lines, time course of probability of occurrence(tau  = 200 ms; original traces shown in Fig. 4, A and B). Note the loss of events occurring at latencies <200 ms in the case of the 60-Hz data. The numbers 1-4 denote the sections of data produced by progressive elimination of early events as shown in inset. B: cumulative amplitude distributions for 4 sections of high-frequency data (thin lines) and low-frequency control. Numbering as in A; asterisks denote significant differences. Actual KS-test P values were 1: <0.0001; 2: 0.004; 3: 0.29; and 4: 0.782. C: comparison as in B for distributions of times to peak. Actual KS-test P values were 1: 0.0004; 2: 0.0178; 3: 0.239; and 4: 0.49. D: correlation between KS statistic (Dmax; i.e., maximal ordinate difference of the cumulative distributions) for the amplitude and time to peak comparisons. Correlation coefficient, 0.99.


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FIG. 6. A m p l i t u d e   d i s t r i b u t i o n s   o fasIPSCs are shifted with changes in probability of release (Pr). A: sample traces (1,260 ms) of evoked responses during high (Aa-Ac) and low (Ad-Af) Pr, controlled by perfusion with extracellular solution containing 2 mM SrCl2:5 mM MgCl2 and 10 mM SrCl2:1 mM MgCl2, respectively. Circles, automatically detected onsets and peaks of individual asIPSCs. Ba: averaged responses (n = 15) from same experiment at low (open triangle) and high (open circle) Pr; closed triangle, average at low Pr scaled to peak amplitude of high-Pr average. Note that average time course was not significantly changed. Bb: latency histograms for asIPSCs detected at high (top panel) and low (bottom panel) Pr. Open portion of the top histogram indicates period not included in the amplitude comparison (see text for details). Thick line is a monoexponential fit. Ca: cumulative amplitude distribution for asIPSC obtained with low (n = 243) and high (n = 804) Pr. Note the rightward shift (KS test; P < 0.0001). Insets: superimposed traces of single asIPSCs recorded under conditions of low and high Pr, respectively. Scale bars, 40 pA and 10 ms. Cb: pooled amplitude distribution of asIPSCs from 5 different cells normalized to mean of low Pr distributions (P < 0.001). Short bars above and below plot indicate the KS 95% confidence intervals (CI) (Van der Kloot 1991). Da: thick lines, cumulative histograms of times to peak from datasets in Ca. Distributions are not significantly different (P > 0.5). Thin line, distribution of times to peak when events in 1st 200 ms of response were included (cf. Fig. 5). Inset: superimposed averages (n = 100) of asIPSCs recorded under conditions of low and high Pr. Scale bars, 10 pA and 20 ms. For comparison of time courses, the low-Pr average is also shown scaled to the peak of the high-Pr average. Db: pooled cumulative frequency distributions for times to peak for 5 experiments with 95% CIs.

Of the experiments using changes in Sr2+:Mg2+ ratio, five of eight fulfilled the criteria of stable series resistance and holding current, absence of rundown of transmission, and good quality of detection. Event frequencies were calculated by dividing the number of detected events by the length of analyzed data traces. We obtained mean frequencies of 4.8 ± 2.0 Hz with 2 mM Sr2+ and 5 mM Mg2+ (low Pr) and 22.1 ± 2.6 Hz after changing the extracellular divalent cation composition to 10 mM Sr2+:1 mM Mg2+ (high Pr). Example traces from one experiment are shown in Fig. 6A. Responses were averaged (Fig. 6Ba) and latency histograms for asIPSCs constructed (Fig. 6Bb) to ascertain that events had occurred in a stimulus-dependent manner under conditions of both high and low Pr. In all experiments, amplitude distributions were shifted toward higher values with high Pr. The cumulative amplitude distributions for the experiment shown in Fig. 6, A and B, as well as superimposed single asIPSCs recorded under both conditions are given in Fig. 6Ca. The amplitude distributions were significantly different (KS test: P < 0.0005) in four cases, whereas in the remaining experiment P = 0.07. The pooled amplitude distributions from all five experiments (normalized to the mean of low Pr values of each dataset) are shown in Fig. 6Cb (KS test: P < 0.001). The comparison of times to peak of the events included in the analysis is shown in Fig. 6Da (KS test: P > 0.5). Figure 6Db shows the pooled, normalized time to peak distributions for the datasets used in analyzing all five experiments (KS test: P > 0.5). The absence of kinetic changes in the asIPSCs that were included in the amplitude comparison is also evident from the comparison of the average asIPSC time course shown in the inset of Fig. 4Da.

The observed rightward shift in the distribution of asIPSC amplitudes in every case was associated with an increase in the coefficient of variation (from an average of 80 ± 17.8% to 95 ± 20.4%) and skew (from 2.2 ± 1.40% to 4.0 ± 0.85%). Because these parameters are insensitive to the corresponding changes in mean amplitude (42.9 ± 14.33 pA to 70.5 ± 29.36 pA), this would not be expected if the altered distribution were due to changes in postsynaptic responsiveness. A change in postsynaptic receptor sensitivity to GABA is further unlikely because the mean decay time course of asIPSCs was unchanged (Fig. 6Da, inset). We also tested for possible changes in postsynaptic receptor sensitivity by making the same changes in extracellular divalent cation composition during steady-state responses to exogenously applied GABA (3 or 10 µM). We found that GABA-induced Cl- currents were reduced to 91.5 ± 4.3% of control by changing the divalent cation ratio from 2 mM Sr2+:5 mM Mg2+ to 10 mM Sr2+:1 mM Mg2+ (n = 4, data not shown). Assuming a sensitivity of the GABAA receptors for intracellular Sr2+ as described in sensory neurons (Behrends et al. 1988), this might be expected for a small leak of Sr2+ into the postsynaptic cell. In fact, a substantial reduction of GABAA responses by physiological concentrations of extracellular Ca2+ was described in sensory neurons (Akaike et al. 1989). A suppressant effect, however, is the reverse of what would be expected if a change in postsynaptic sensitivity were to explain the observed rightward shift in asIPSC-amplitude distributions.

Physiologically, Pr at many synapses (including GABAergic contacts in the striatum) is decreased by presynaptic inhibition through GABAB receptors (Nisenbaum et al. 1992; Verhage et al. 1994), due at least in part to a decrease in Ca2+-channel activity (Pfrieger et al. 1994). We therefore used the GABAB-receptor agonist baclofen (3-10 µM) to produce a state of low Pr that may occur under physiological conditions (Fig. 7, A-C). In agreement with the findings of Nisenbaum et al. (1992) in the slice preparation, baclofen did not induce detectable postsynaptic conductance changes but lowered the mean frequency of asIPSCs from 7.0 ± 1.3 Hz to 1.7 ± 0.1 Hz (n = 4 experiments that fulfilled criteria out of a total of 6) when the extracellular solution contained 2 mM Sr2+:1 mM Mg2+. In all four cases, amplitude distributions of asIPSCs were significantly shifted toward lower values (Fig. 7G; KS test: P < 0.0005) without changes in the distribution of times to peak (P > 0.5) or in the time course of asIPSCs (Fig. 7, D-H). The mean amplitudes changed from an average of 47.4 ± 12.37 pA to 32.17 ± 12.32 pA, and the coefficient of variation of the distributions decreased from an average of 77 ± 7.5% to 68 ± 3.1%. The skewness of the distributions increased slightly from 2.3 ± 0.19 to 2.8 ± 0.13. 


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FIG. 7. A m p l i t u d e   d i s t r i b u t i o n s   o fasIPSCs are shifted to the left by gamma -aminobutyric acid-B (GABAB)-receptor-mediated presynaptic inhibition. A: superimposed averages of postsynaptic asIPSCs (1,260 ms) under control conditions (2 mM Sr2+:1 mM Mg2+) and following application of baclofen at 5 µM. The latter average is also shown scaled to control peak amplitude. Note unchanged time course of the average IPSC. Upward arrow, end of period where asIPSC frequency was too high for reliable detectionunder control conditions (300 ms; seeMETHODS). B and C: sample traces of responses recorded under control conditions (shown from 300-1,260 ms after onset) and in the presence of baclofen (10-970 ms), respectively. Circles, detected onsets and peaks of asIPSCs. D: averaged (n > 100) asIPSCs detected in control period and after baclofen application. Note the decrease in mean amplitude. Latter average is also drawn scaled to control peak, illustrating that the waveform of asIPSCs was not affected. E and F: superimposed single asIPSCs (n = 50) recorded under control conditions and in presence of baclofen, respectively. G: pooled, cumulative distributions of amplitudes (normalized to control means) from 4 experiments (P < 0.0005). Short bars above and below line show 95% CI. F: distributions of times to peak corresponding to the amplitude distributions in G (P > 0.5).


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FIG. 8. L a t e n c y - d e p e n d e n t   s h i f t   i nasIPSC-amplitude distributions. Aa: superimposed asynchronous responses recorded in 2 mM Sr2+:1 mM Mg2+. Ab: poststimulus time histogram for this experiment, fitted with a single exponential (thick line). Upward arrow, time bin of initial frequency (40 ms; see text); downward arrow, time bin by which frequency has fallen to 1/e (180 ms). Ba: cumulative amplitude distributions of asIPSCs occurring before (early; n = 219) and after (late; n = 131) the frequency had declined to 1/e. Note shift toward lower amplitudes for later events(P < 0.001). Inset: superimposed averages of early and late asIPSCs. Note the decrease in mean peak amplitude. Average of the late asIPSCs is also shown scaled to allow kinetic comparison. Bb: cumulative distributions of times to peak of early and late events; distributions are not significantly different (P > 0.5). Ca and Cb: pooled, cumulative amplitude (P < 0.01) and time to peak distributions (P > 0.1), respectively, from all 7 experiments, normalized to the mean values of early asIPSCs.

Although to our knowledge it has not been suggested that GABAB-receptor-activated intracellular signaling pathways act on GABAA-receptor-mediated responses, we tested for possible effects of baclofen on the sensitivity of GABAA receptors. Baclofen in concentrations between 3 and 10 µM was without any measurable effect on steady-state currents evoked by exogenous GABA (3 or 10 µM, n = 5; data not shown).

Evidence for latency-dependent changes in quantal-size distributions

asIPSCs occurring later in a response would be expected to originate from synapses that have lower Pr than those responding earlier, either because intraterminal Sr2+ concentration by then has decayed to lower levels or because, assuming a continuum of intrinsic release probabilities (Murthy et al. 1997), intrinsically low Pr synapses release later than those with intrinsically high Pr. The relationship between Pr and quantal size suggested by the above experiments should then produce a leftward shift in the distribution of quantal sizes with latency. To address this issue, we constructed separate sets of amplitude data from seven pairs (recorded in 2 mM Sr2+:1 mM Mg2+) for asIPSCs occurring before and after the point in time where the frequency of events had fallen to 1/e of its initial value. The initial frequency was determined as that in the first 20-ms bin where events were detected reliably. Figure 8, A and B, illustrates an experiment in which the amplitude distribution of early events was significantly shifted toward larger amplitudes with respect to that of later events (KS test: P < 0.001; Fig. 8Ba), whereas the distribution of times to peak was not affected (Fig. 8Bb). The later findings indicates as before, that the shift in amplitude distribution was not produced by enhanced collision probabilities (see Changes in Pr affect asIPSC-amplitude distributions). The same result was obtained in four of seven experiments. The P value for the KS comparison of pooled, normalized amplitude data for all seven pairs (Fig. 8Ca) was <0.01, which does not allow us to conclude that the two distributions are identical. Following a suggestion of Van der Kloot (1991), we also performed an analysis of variance (ANOVA) on these pooled data following logarithmic transformation (Bonferroni-Dunn,P < 0.0001). The times to peak were not significantly different in the two pooled distributions (KS test: P > 0.1;Fig. 8Cb).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Asynchronous evoked inhibitory transmission in presence of Sr2+

We have recorded asIPSCs evoked by stimulation of a single presynaptic cell in the presence of extracellular Sr2+ instead of Ca2+. The time courses of these events were indistinguishable from those of mIPSCs, indicating that they represent responses to single release events originating at individual synaptic contacts. Desynchronization by Sr2+ of evoked transmission into single quantal events has been described at the neuromuscular junction (Bain and Quastel 1992; Dodge et al. 1969; Meiri and Rahamimoff 1971; Zengel and Magleby 1980) as well as at hippocampal excitatory synapses (Abdul-Ghani et al. 1996; Goda and Stevens 1994; Oliet et al. 1996). Our results show that this effect is also present at inhibitory connections. Presently, the mechanism of this desynchronization has not been fully elucidated. The most parsimonious explanation may be to attribute it to a slow (with respect to Ca2+) binding of the Sr2+ ions to the release-inducing site (cf. Fig. 1) and to a slow removal of intracellular Sr2+ (Bain and Quastel 1992). Thus we assume that the asynchronous response produced by Sr2+ is simply due to dispersion in time of the process responsible for evoked transmitter release (cf. Abdul-Ghani et al. 1996). Alternatively, by analogy with the selective abolition of synchronous release in synaptotagmin knockouts (Geppert et al. 1994), it has been suggested that a special mechanism for asynchronous release is preferentially activated by Sr2+ ions (Goda and Stevens 1994). However, because it has not been shown that Sr2+-evoked asynchronous release is intact in these mutant synapses, direct evidence for this hypothesis is lacking.

Quantal variance and probability of release

The coefficient of variation of the skewed asIPSC-amplitude distributions was equal to or larger than that of mIPSCs. Therefore, as at excitatory synapses, the responses to individual release events originating from the contacts of a single presynaptic cell are not more homogeneous in amplitude than those originating from the entire population of contacts made on the postsynaptic cell (Oliet et al. 1996).

In the setting of the all-or-nothing hypotheses, the skewed, highly variant distribution of quantal sizes has been exclusively attributed either to the variable volume and, therefore, transmitter content of vesicles (Bekkers et al. 1990; Bekkers and Stevens 1995; Frerking et al. 1995) or to variation from site to site according to the number of postsynaptic receptor clusters present (Edwards 1995; Edwards et al. 1990; Jonas et al. 1993; Kullmann 1993). The latter hypothesis implies saturation of postsynaptic receptors facing a given release site and predicts that the variance associated with responses generated at a single site is low. However, there is increasing evidence that the amplitude variance of synaptic currents elicited or recorded from single or a few boutons is comparable with the variation in amplitude of population miniatures (Bekkers et al. 1990; Bekkers and Stevens 1995; Lewis and Faber 1996; Liu and Tsien 1995), suggesting that at many synapses each contact has a wide repertoire of quantal sizes.

The main finding of the present study is that quantal size distributions, as estimated from the amplitudes of asIPSCs, are shifted in the same direction as the frequency of release by manipulations that change Pr. This effect cannot be explained by changes in postsynaptic receptor sensitivity, and appropriate precautions were taken against artifactual changes in amplitude distributions with frequency that may occur by superposition of single events (see RESULTS and Figs. 4 and 5).

An increase in quantal size with Pr requires an increase in the amount of transmitter released as well as an enhanced occupancy of postsynaptic receptors facing a presynaptic contact. Synchronized release of multiple vesicles (Tong and Jahr 1994), either from a single or from multiple neighboring release sites can thus augment quantal size under the condition that receptors are not already saturated by the smallest releasable amount of transmitter (Otis et al. 1996; Silver et al. 1996). Receptor saturation is likely at some synapses but not at others (for review see Frerking and Wilson 1996). Recently, Nusser et al. (1997) showed evidence strongly suggesting that receptor occupancy during mIPSCs is heterogeneous in cerebellar granule cells and that it depends on the number of postsynaptic receptors present. Coincident multivesicular release was also invoked in studies of miniatures when equidistant peaks were apparent in amplitude distributions (Lewis and Faber 1996; Paulsen and Heggelund 1996). The same conclusion was reached by Poisbeau et al. (1996), who observed a change in mIPSC-amplitude distribution with decreases in extracellular Ca2+ concentration, and also by Vincent and Marty (1996) to explain the large fluctuations in amplitudes of evoked IPSCs. It is noteworthy that equidistant peaks in quantal amplitude distributions are only to be expected if responses to single vesicle releases sum linearly, which they are likely to do only if contacts operate in a linear range of the concentration response curve for the transmitter and if other sources of variation (such as vesicle sizes and channel gating) do not smooth out the distribution. Therefore amplitude distributions without obvious peakedness such as shown here do not rule out multivesicular release. The degree of synchronization of multiple vesicles would depend on the intensity of the intraterminal Sr2+ signal. Similar to the model proposed by Goda and Stevens (1994), there might be a set of high-affinity binding sites that trigger release of single vesicles, whereas a population of lower-affinity sites also would have to be occupied to produce concerted exocytosis of several vesicles. However, cooperative interactions between occupied binding sites or primed vesicles (Van der Kloot and Molgò 1995) might also explain this effect without assuming multiple binding sites for transmitter release.

As an alternative, it was recently suggested that synaptic vesicle fusion may not be a catastrophic event but rapidly reversible before the content of a vesicle is fully discharged into the synaptic cleft (Rahamimoff and Fernandez 1997). This scheme equally provides a link between Pr and quantal size, because the amount of transmitter released depends on the time for which the vesicle lumen remains connected to the intrasynaptic space (Clements 1996). This "vesicle open time" may then become a correlate of Pr, as it would depend on the balance between the rates of reactions that stabilize and those that reverse fusion. Given the fact that the fusion reaction depends on multiple interactions between proteins (Südhof 1995), the distribution of vesicle open times might be a kind of survival function, such as the gamma distribution (Pitman 1993), which can also be used to approximate quantal size distributions (Fig. 2C) (McLachlan 1978). Both multivesicular release and regulated postfusion release would fit the result that quantal size distributions change with Pr.

Miniature events versus asynchronous evoked responses

Presynaptic changes in synaptic efficacy are traditionally associated with selective changes in the frequency of quantal events recorded as mIPSCs. Although some published data are at variance with this view (see below), it is customary to regard the absence of alterations in miniature amplitude distributions as a criterion for presynaptic effects on synaptic transmission. In a number of reports regarding presynaptic modulation at inhibitory synapses, this stipulation was found to hold (Behrends and ten Bruggencate 1993; Capogna et al. 1993, 1995, 1996; Doze et al. 1995; Le Feuvre et al. 1997; Thompson et al. 1993; Trudeau et al. 1996).

However, there are important differences between the asIPSCs studied here and mIPSCs. First, with IPSCs the population of active synapses is limited to those emanating from a single presynaptic neuron, so that a relatively greater number of events is sampled from a given contact and the estimate of the quantal size distribution should become more reliable. For the same reason, compared with miniatures, the contribution of quantal variance at a single contact to the shape of the measured distribution is enhanced with respect to the impact of between-site variance. Selective activation of contacts from one presynaptic neuron also reduces the likelihood that the population of active sites changes dramatically when Pr is altered. Such population shifts (e.g., silencing or recruitment of a subset of contacts with decreases or increases in Pr, respectively) may, in the measured distribution, obscure changes in quantal size at single sites. In other words, it is uncertain with miniatures whether the change in frequency observed is indeed due to a change in Pr at previously active sites or whether the predominant change was in the number of active contacts.

Second, miniature events reflect release activity in the absence of stimulation (i.e., in the lowest range of Pr at the individual contacts). This is especially evident in cases where miniature frequency is unaffected by reducing extracellular Ca2+ concentration or by blocking Ca2+ channels (e.g., Llano and Gerschenfeld 1993; Scanziani et al. 1992; Scholz and Miller 1992). In contrast, asIPSCs are caused by a phasic increase in release activity, which is closer to normal action potential-evoked release. A further possible reason for the discrepancy of our findings with the miniature studies cited above is therefore that contacts make use of their full quantal size repertoire (e.g., synchronously release more than 1 quantum) only when release activity is rapidly increased well above its constitutive level. Interestingly, some studies have shown that mIPSC frequency does depend on extracellular Ca2+ (Kraszewski and Grantyn 1992; Poisbeau et al. 1996), suggesting that an important fraction of events was due to random openings of presynaptic Ca2+ channels and that release occurred because Pr was transiently and rapidly elevated by a rise in intraterminal Ca2+ concentration. As expected from this reasoning, these reports also show a clear consensual shift with frequency of mIPSC-amplitude distributions in response to a change in extracellular Ca2+ concentration.

On the other hand, Doze et al. (1995) observed no change in mIPSC-amplitudes when presynaptic terminals were tonically depolarized by application of high potassium solutions, although their frequency did rise markedly. In addition, in this situation mIPSC frequency was reduced but amplitudes were unchanged by partial block of Ca2+ channels or by baclofen application (Doze et al. 1995; Momiyama and Takahashi 1994). Similar results were obtained by Capogna et al. (1996) when release activity was tonically enhanced by either the Ca2+-ionophore ionomycin or the direct secretagogue alpha -latrotoxin. However, the increase in mIPSC frequency produced by such measures is evidently not equivalent to release activated by a phasic Sr2+ influx, as studied in the present report. Also, with tonic activation of release the problems inherent in the estimation of quantal size with miniature recordings, such as shifts in the population of active contacts, become aggravated. Furthermore, vesicles or cofactors necessary for release may be depleted during such long-term enhancement of release and thus preclude a presynaptic enhancement of quantal size.

More directly, our present findings appear to contradict a recent study at hippocampal excitatory synapses in the slice preparation (Oliet et al. 1996). These authors used paired-pulse stimulation to facilitate Sr2+-dependent asynchronous transmitter release and failed to observe a shift in quantal size distributions. Because extracellular stimulation was used, it is likely that a large number of different presynaptic neurons was activated in their experiments. A shift in the population of active contacts (e.g., enhanced recruitment of intrinsically low Pr synapses) (cf. Hessler et al. 1993; Murthy et al. 1997; Rosenmund et al. 1993) that might have balanced an increase in quantal size at previously active sites can, therefore, not be excluded. However, it is equally possible that differences in vesicular content between synapses account for this discrepancy.

It is important to note here that the findings of our present study cannot easily be explained by a shift in the active population of contacts with fixed quantal sizes. Such an explanation would require that contacts with a fixed quantal size above the average (i.e., larger vesicles or more postsynaptic receptors) had lower than average intrinsic Pr, so that on changing the overall release activity this subset is either included into or excluded from the active population, thus producing shifts in the amplitude distribution of asIPSCs. The leftward shift in asIPSC-amplitude distributions we observed for later (as compared with earlier) events in an asynchronous response, however, suggests, that if heterogeneity is present regarding intrinsic Pr and quantal size among synapses of one presynaptic neuron, larger quantal events are more likely to be contributed by contacts with intrinsically higher, not lower, Pr. This follows from the fact that contacts with high intrinsic Pr will tend to release early and is in line with our interpretation that quantal size is positively related to Pr as well as with recent findings at excitatory synapses suggesting that transmission at larger contacts with more vesicles is more reliable (Murthy et al. 1997). The latency dependence of quantal sizes we observed also suggests that because we often had to disregard the earliest period of release when activity was too dense for reliable detection of events, we probably underestimated the range of quantal amplitudes under conditions of high Pr. Therefore the measured changes in quantal size distributions with Pr are likely to be somewhat underestimated as well.

In summary, the data presented here provide evidence that at GABAergic synaptic contacts between striatal neurons in cell culture, quantal size measured as amplitudes of asIPSCs in the presence of extracellular Sr2+ can be modified through experimental changes in Pr, including activation of a physiologically important presynaptic modulatory pathway via the GABAB receptor. These findings introduce the possibility that, in contrast to predictions of the all-or-nothing hypotheses, presynaptic modulation could locally influence the weight of synaptic inputs. Whether this degree of freedom for information transfer at single contacts exists at other synapses as well, or, indeed, whether it exists at synapses made by striatal neurons in the normally differentiated mammalian brain, will have to be ascertained by further studies.

    ACKNOWLEDGEMENTS

  We thank N. Ankri for the event detection software; N. Tokutomi for the gift of a Y-tube application system; and L. Penner, L. Kargl, and T. Simon-Ebert for excellent technical assistance. We are grateful to N. Ankri for helpful discussions; G. W. Kreutzberg and the Max Planck Institute for Psychiatry, Martinsried, for supporting us with equipment; and to L. Sachs for the clarification of statistical issues.

  This work was supported by Deutsche Forschungsgemeinschaft Grant Be-1739/1 and the Friedrich Baur-Stiftung.

    FOOTNOTES

  Address for reprint requests: J. C. Behrends, Physiologisches Institut der Universität München, Pettenkoferstr. 12, 80336 Munich, Germany.

  Received 4 September 1997; accepted in final form 22 January 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society