Sr2+-dependent asynchronous evoked transmission at rat striatal inhibitory synapses in vitro

Abstract

1. At striatal inhibitory synapses in cell culture, replacement of extracellular Ca2+ with Sr2+ desynchronized inhibitory postynaptic currents (IPSCs), reducing their peak amplitude and producing a succession of late, asynchronous synaptic events (late release). In the averaged IPSC waveform this resulted in an increase in both the fast and the slow decay time constant as well as in the time to peak. 2. Rapid removal of extracellular Sr2+ during late release was without effect on the time course of the averaged IPSC. Thus, late release is not dependent on continuous Sr2+ influx, but must be related to the way in which Sr2+, as opposed to Ca2+, interacts with constituents of the intracellular space. 3. After application of the membrane-permeant acetoxymethyl ester (AM) form of the Ca2+-chelator BAPTA, Sr2+-induced late release was greatly reduced and the kinetics of the Sr2+-dependent IPSC approached those of the Ca2+-dependent response. EGTA AM had a similar but less pronounced effect. 4. Using rapid solution exchange, we stimulated synapses first in Sr2+- or Ca2+- and 100-300 ms afterwards in Ca2+-containing solution. Paired-pulse facilitation of late release was the same whether the conditioning pulse induced a presynaptic influx of Sr2+ or of Ca2+. 5. It is concluded that Sr2+-mediated asynchrony is probably due to a less efficient intraterminal buffering of Sr2+ as opposed to Ca2+, allowing for Sr2+ ions to activate release in an area less confined to the immediate vicinity of the presynaptic Ca2+ channel. This hypothesis explains both the action of endogenous buffers and the apparent lack of specific facilitatory interaction between Ca2+-mediated and Sr2+-induced late release.

Figure 1. Sr²⁺ substitution desynchronizes GABAergic transmission

A and B, superimposed traces show IPSCs (n= 5) recorded in response to a presynaptic action potential in the presence of 2 mM Ca²⁺(A) or 2 mM Sr²⁺(B). Insets: the same responses displayed at an expanded time scale. Note the massive desynchronization of the response in Sr²⁺. C, superimposition of the averages (n= 15) of IPSCs recorded in Ca²⁺ and Sr²⁺. Inset: averages shown scaled to peak and at an expanded time scale. Note the longer time to peak of the Sr²⁺ response. Da, time course of the integrals of the averaged IPSCs recorded in Ca²⁺ (▪) and Sr²⁺ (•). Error bars: standard error of the mean. In this example, the mean total synaptic charge flowing in Sr²⁺ exceeds that transferred during the Ca²⁺-dependent response. Note however, that due to the large variance of integrals the difference between the two final values is not statistically significant (11.62 ± 1.76 pC vs. 17.30 ± 3.79 pC, P > 0.05). Db, time courses of the peak-normalized integrals, obtained by dividing the integrals in a by the peak amplitude of the response (see Methods). □, Ca²⁺; ○, Sr²⁺. The difference between the final values is highly significant (51.16 ± 0.77 ms vs. 182.14 ± 39.95 ms, P < 0.005).

Figure 2. Rapid removal of Sr²⁺ does not affect the kinetics of Sr²⁺-mediated late release

A, validation of the rapid solution exchange using bicuculline. IPSCs were elicited by extracellular stimulation in 10 mM Sr²⁺. Aa, stimuli were repeatedly applied at different intervals with respect to the onset of a 250 ms rapid perfusion of the cell with bicuculline (10 μm)-containing solution (filled bar). Note the decrease of the peak amplitude of responses elicited in the time window of bicuculline perfusion. Ab, Sr²⁺-mediated IPSCs were elicited and averaged (n= 15) with or without the perfusion of bicuculline-containing solution for 250 ms starting 50 ms after the stimulus. Intervals of 5 s were allowed between stimuli. Averaged responses are shown superimposed and scaled to peak to enable comparison of kinetics. A clear deviation in time course due to the action of the antagonist becomes visible almost immediately after activation of the switch. The difference trace shown above the dashed line clearly illustrates the onset and offset of bicuculline action. Data in Aa and b are from the same neurone. B, rapid replacement of 10 mM Sr²⁺ by 10 mM Mg²⁺. Ba, protocol similar to Aa: the stimulus was repeatedly given at varying intervals with respect to the onset of rapid perfusion of Sr²⁺-free high-Mg²⁺ solution for 400 ms. Moving the stimulus into the Mg²⁺ perfusion time window (filled bar) leads to a progressive loss of synaptic current, indicating that Sr²⁺ is being washed out rapidly. Bb, protocol similar to Ab: superimposition of averaged, scaled (n= 15) Sr²⁺-mediated IPSCs elicited with or without a rapid switch from Sr²⁺- into Mg²⁺-containing solution 50 ms after the stimulus. Note the absence of any difference in the time course. Data in Ba and Bb are from the same neurone.

Figure 3. Comparison of Sr²⁺ and Ca²⁺ binding to BAPTA

Increasing amounts of Ca²⁺ and Sr²⁺, respectively, were added to two aliquots of 100 μm BAPTA (filled symbols). In a control experiment, the same comparison was made for Sr²⁺ and Mg²⁺ (open symbols). Each data point is the mean of two or three repetitive absorption measurements at a wavelength of 255 nm. s.d. < symbol size. The data set shown here represents one of two independent experiments which gave identical results.

Figure 4. Application of BAPTA AM suppresses Sr²⁺-mediated late release

A, superimposed peak-scaled traces show the differential effect of BAPTA AM on the kinetics of IPSCs evoked in 2 mM Ca²⁺ and in 5 mM Sr²⁺. The Ca²⁺-mediated response is kinetically unaffected (a). Sr²⁺ substitution induces the characteristic asynchrony of the response (b). Note, however, that hardly any difference is detectable between the response recorded in 2 mM Ca²⁺ and that obtained in 5 mM Sr²⁺ after application of BAPTA AM (c). B, semilogarithmic plot of the responses shown in A, illustrating the changes in time constants. Note the return to control of the fast time constant of the Sr²⁺-mediated response after application of BAPTA AM. C, summary of the results of 7 experiments (see text for details). All of 7 cell pairs were tested in all 4 conditions.

Figure 5. Effect of EGTA AM on Sr²⁺-mediated late release

A, the effect of EGTA AM is shown as in Fig. 4A for BAPTA AM. Note the reduced but persistent asynchrony after application of EGTA AM (c). B, as shown for BAPTA AM in Fig. 4B. Note: the fit for 5 mM Sr²⁺ (○) appears as a straight line because early and late time constants are closely similar. This occurs when late release becomes dominant. When the early decay component becomes faster under the influence of EGTA AM (□) the difference between fast and slow exponentials is unmasked. In this case, there is also an apparent increase in the time constant of the latter. C, as shown in Fig. 4C for BAPTA AM. Note the significant increase in the normalized integral by Sr²⁺ substitution despite application of EGTA AM.

Figure 6. Paired-pulse facilitation of late release is not increased by a conditioning pulse in Sr²⁺

A, set-up of the rapid perfusion device: IPSCs recorded either in Sr²⁺-containing solution or after a rapid switch into Ca²⁺ saline. Note the clear difference in kinetics. Ba-c, averaged IPSCs (n > 5) with conditioning and test pulse delivered in the solutions indicated. Dashed lines: extrapolated time courses of conditioning IPSCs. Insets: superimposed time course of first response (I) and second response (II) in each sequence. Note that facilitation of late release occurred in all cases except the Sr²⁺, Ca²⁺ sequence. C, superimposed, isolated test IPSCs (see text) resulting from the three sequences. Note the absence of any difference between the kinetics of test IPSCs of the Ca²⁺, Ca²⁺ and Sr²⁺, Ca²⁺ sequence.