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. 2003 Nov 19;23(33):10475-86.
doi: 10.1523/JNEUROSCI.23-33-10475.2003.

Regulation of exocytosis from single visualized GABAergic boutons in hippocampal slices

Darrin H Brager  1 , Paul W Luther, Ferenc Erdélyi, Gabor Szabó, Bradley E Alger
Affiliations

Regulation of exocytosis from single visualized GABAergic boutons in hippocampal slices

Darrin H Brager et al. J Neurosci. .

Abstract

Regulation of GABA release is crucial for normal brain functioning, and GABAA-mediated IPSCs are strongly influenced by repetitive stimulation and neuromodulation. However, GABA exocytosis has not been examined directly in organized tissue. Important issues remain outside the realm of electrophysiological techniques or are complicated by postsynaptic factors. For example, it is not known whether all presynaptic modulators affect release from all boutons in the same way, or whether modulator effects depend on the presence of certain types of voltage-gated calcium channels (VGCCs). To address such issues, we used confocal imaging and styryl dyes to monitor exocytosis from identified GABAergic boutons in organotypic hippocampal slice cultures. Repetitively evoked IPSCs declined more rapidly and completely than exocytosis, suggesting that depletion of filled vesicles cannot fully account for IPSC depression and underscoring the usefulness of directly imaging exocytosis. Stimulation at 10 Hz produced a transient facilitation of exocytosis that was dependent on L-type VGCCs. Using specific toxins, we found that release mediated via N-type and P-type VGCCs had similar properties. Neither baclofen nor a cannabinoid receptor agonist, CP55940, affected all boutons uniformly; they slowed release from some but completely prevented detectable release from others. Increasing stimulus frequency overcame this blockade of release. However, baclofen and CP55940 did not act identically, because only baclofen reduced facilitation and affected bouton releasing via P/Q-type VGCCs. Direct observation thus revealed novel features of GABAergic exocytosis and its regulation that would have been difficult or impossible to detect electrophysiologically. These features advance the understanding of the regulation of synapses and networks by presynaptic inhibition.

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Figures

Figure 1.
Figure 1.
Loading of GABAergic boutons with styryl dye. A, Protocol for loading organotypic hippocampal slices with Synaptogreen-C4 dye. The dye was present in the slice for 2 min before field stimulation of 10 Hz for 3 min was given and for 2 min after stimulation to allow for uptake. The dye was removed and the chelator ADVASEP-7 was perfused for ∼20 min before an unloading stimulus train was given. B, Boutons loaded in s. pyamidale and s. radiatum in the absence and presence of adenosine. Scale bar, 20 μm. C, Group data showing numbers of labeled boutons present in s. radiatum and s. pyramidale in the absence (control) or presence of 50 μm adenosine. There was a highly significant reduction in the numbers of boutons in s. radiatum and no change in s. pyramidale. *, Significantly different from control; #, significantly different from s. pyramidale. D, Distribution of the sizes (areas) of 834 labeled boutons from nine rat slices in s. pyramidale. E, Overlay image of Synaptored-loaded boutons in GAD65-eGFP mouse culture. Arrowheads in E point to Synaptored-labeled boutons in a GAD65-eGFP mouse culture. Arrows in inset indicate yellow clusters of Synaptored-filled vesicles with eGFP-labeled boutons. Scale bar, 10 μm. F, Distribution of Synaptored-loaded bouton sizes of 255 boutons from three slices from GAD65-eGFP mice.
Figure 2.
Figure 2.
Repetitive stimulation causes destaining of boutons. A, Images taken before stimulation, 15 sec after beginning stimulation, and 1 min after the end of stimulation. Arrowheads point to Synaptogreen-labeled boutons in a rat culture. B, Destaining profiles for 50 boutons from a single slice caused by 5 Hz field stimulation. Each trace is from a single bouton imaged at successive 15 sec intervals throughout the trial (nondestaining boutons are indicated in red). C, Mean data from the individual traces in B.
Figure 3.
Figure 3.
Frequency dependence of destaining. A, Group data showing destaining induced by different field stimulation frequencies. The numbers of boutons in each of the 14 slices (2 Hz, 5; 5 Hz, 3; 10 Hz, 6) represented ranged from 33 to 147 (mean, 78 ± 10; different slices per frequency; no significant difference in numbers of loaded boutons between slices). B, The mean time constant of destaining is frequency dependent. The symbols represent the mean time constants of decay at the indicated frequencies obtained from the best fits of exponential curves to the data in A. The solid line is the fit of the data by an empirical equation, analogous to that used by Zakharenko et al. (2001), to illustrate the kinetics of destaining of boutons in CA1 s. radiatum: τdestain = τ(min) + q/f, where τdestain is the time constant of destaining, τ(min) is the minimal time constant at 10 Hz, f is the frequency of stimulation, and q is a fit constant. C, The steady-state level of fluorescence at the end of stimulation trains declines as the frequency of stimulation during the trains goes up. The numbers of nondestaining boutons remained constant across different frequencies of stimulation. D, Destaining data from each bouton were fit by single exponentials. The bar graph plots the distribution of the time constants for the indicated numbers of boutons that made up the groups in A (total, n = 797 boutons). Note the shift in the population means (arrows) to faster time constants as the frequency increases. E, Cd2+ prevents destaining. The filled circles represent fluorescence measurements from 154 boutons in three slices that were loaded, washed, and imaged normally but received no unloading stimulation. The slight decrease in fluorescence by the end of the trial is the result of photo-bleaching. Data represented by open circles were obtained from measurements of 159 boutons in three slices loaded in normal solution, washed for 20 min in ADVASEP-7 plus 100 μm Cd2+, and then stimulated at 5 Hz (black bar) still in the presence of Cd2+. Cd2+ was then washed from the chamber for ≥20 min, and the slices were stimulated again (open diamonds). Cd2+ essentially prevented destaining (cf. open circles and diamonds); the data in Cd2+ are not significantly different from the photo-bleaching data.
Figure 4.
Figure 4.
Comparison of repetitive stimulation on dye release and monosynaptic evoked IPSCs at 2 and 10 Hz. A, Decrease in IPSC amplitudes (from 3 cells, 3 slices) caused by 150 stimuli delivered at 2 Hz (•) or 10 Hz (○). Inset, Representative IPSCs, means of 3 each, at the indicated times. Calibration: 200 pA, 25 msec. B, Comparison of the destaining of Synaptogreen-loaded boutons (•) and the depression of IPSCs (○) during 2 Hz stimulation. Fluorescence measurements were made after the indicated number of stimuli. For IPSC depression, the last two IPSCs in the indicated period were averaged (see Results). C, Comparison of the destaining of Synaptogreen-loaded boutons and IPSC depression after 15 sec of 10 Hz stimulation.
Figure 5.
Figure 5.
Fractional destaining of GABAergic boutons. The fractional destaining rate, f (see Materials and Methods), is an estimate of the average amount of release induced by an action potential in a given interval. A, Top graphs show group data for f as a function of stimulus number. f remained constant throughout 2 Hz stimulation (left) but transiently increased with 10 Hz stimulation (right). The stimulus trains in A1 started at t = 0; thus, the first f value that could be obtained is after 15 sec of stimulation. To compare f after equal numbers of stimuli, f was calculated after 150 and 300 stimuli for the individual boutons that made up the group data in these plots. Representative data from two slices are shown replotted in A2, where each line represents an individual bouton. Most individual values of f increase (mean f = 0.52) between 150 and 300 stimuli with 10 Hz stimulation (n = 112 boutons) but not with 2 Hz stimulation (mean f = 0.19; 63 boutons). The filled circles represent the means of the groups. B, Group data (n = 4 slices; 278 boutons) showing effects of 10 μm nifedipine on destaining induced by 10 Hz stimulation in the presence of agatoxin. Data in C are from 101 individual boutons (○) and the mean of those boutons (•) from a single slice showing that the transient enhancement of f is blocked (mean f = 0.22) by nifedipine. Graph in the bottom right shows that nifedipine (○) blocked the transient enhancement of f. Control data (•) are replotted from A (bottom) for comparison.
Figure 6.
Figure 6.
Baclofen reduces vesicular release. A, Left, Stimulation at 2 Hz induced IPSCs before (•) and after application of 10 μm baclofen (○). The solid (Fig. 4A, control) and dashed (baclofen) lines represent the steady-state level of IPSC depression computed as the average of the last 10 responses. A, Right, Same as left but with 10 Hz stimulation. Inset shows representative IPSCs (means of 3 each). B, Comparison of control group data (reproduced from Fig. 3D) from slices pretreated with 250 nm agatoxin and four slices (265 boutons) treated first with 10 μm baclofen and then with baclofen plus the GABAB antagonist, CGP55485 (CGP), at 10 μm. C, Baclofen significantly increased the number of nondestaining boutons compared with control and CGP. D, Baclofen significantly shifted the distribution of individual bouton destaining time constants (τdestain) toward longer values, an effect that was also prevented by CGP55485. Arrows indicate the means of the distributions. E, Comparison of control group data from slices pretreated with 250 nm conotoxin and six slices (290 boutons) treated first with 10 μm baclofen and then with baclofen plus CGP55485 at 10 μm. F, Baclofen significantly increased the number of nondestaining boutons compared with control and CGP55485. G, Baclofen had no significant effect on the distribution of τdestain. Stimulation is indicated by the black bar in B and E.
Figure 7.
Figure 7.
The cannabinoid CB1 receptor agonist, CP55940, inhibits vesicular release evoked with 2 Hz stimulation. A, Comparison of control group data (Fig. 3D) from slices pretreated with 250 nm agatoxin and five slices (384 boutons) treated first with 1 μm CP55940 and then with CP55940 plus the CB1 antagonist, AM251, at 4 μm. CP55940 slowed the release elicited by 2 Hz stimulation, and this was prevented by AM251. B, CP55940 increased the percentage of nondestaining boutons over control levels. AM251 plus CP55940 treatment prevented the increase in numbers of nondestaining boutons. C, For the boutons that did destain in CP55940, the distribution of τdestain was shifted toward longer values, an effect that was also prevented by AM251. CP55940 had no effect on mean destaining (D), number of nondestaining boutons (E), or τdestain (F) in four conotoxin-treated slices (136 boutons). Control data are from Figure 6G. Stimulation is indicated by the black bar in A and D.
Figure 8.
Figure 8.
Regulation of GABA release elicited by 10 Hz stimulation. A, Agatoxin-pretreated slices. A comparison of control group data (Fig. 3A) with four slices (324 boutons) also treated with baclofen is shown. B, Baclofen does not increase the percentage of nondestaining boutons when 10 Hz stimulation is given. C, Baclofen shifts the distribution of individual destaining time constants toward longer values, although much less than for 2 Hz stimulation. D-F, Same as for A-C except on three conotoxin-pretreated slices (120 boutons). Stimulation is indicated by the black bar in A and D. G, The transient enhancement of f induced by 10 Hz stimulation is prevented by baclofen, although not by CP55940. Data are plotted for f values after 150 and 300 stimuli as in Figure 5A2. f increases in control conditions (control data calculated from data shown in Fig. 5A2, right plot; agatoxin present). H, Enhancement of f normalized to initial value for each condition.
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