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. 1997 Apr 15;17(8):2738-45.
doi: 10.1523/JNEUROSCI.17-08-02738.1997.

Nonuniform distribution of Ca2+ channel subtypes on presynaptic terminals of excitatory synapses in hippocampal cultures

C A Reid  1 , J D Clements, J M Bekkers
Affiliations

Nonuniform distribution of Ca2+ channel subtypes on presynaptic terminals of excitatory synapses in hippocampal cultures

C A Reid et al. J Neurosci. .

Abstract

Several subtypes of Ca2+ channel support the release of glutamate at excitatory synapses. We investigated the pattern of colocalization of these subtypes on presynaptic terminals in hippocampal cultures. N-type (conotoxin GVIA-sensitive) or P/Q-type (agatoxin IVA-sensitive) Ca2+ channels were blocked selectively, and the reduction in transmitter release probability (Pr) was measured with MK-801. The antagonists completely blocked release at some terminals, reduced Pr at others, and failed to affect the remainder. In contrast, nonselective reduction of presynaptic Ca2+ influx by adding Cd2+ or lowering external Ca2+ reduced Pr uniformly at all terminals. We conclude from these results that the mixture of N-type and P/Q-type channels varies markedly between terminals on the same afferent. The distribution of Ca2+ channel subtypes was the same for high and low Pr terminals. Given that Ca2+ channel subtypes are affected differentially by neuromodulators, these findings lead to the possibility of terminal-specific modulation of synaptic function.

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Figures

Fig. 6.
Fig. 6.
A simple model of a nonuniform distribution of presynaptic Ca2+ channel subtypes accounts for our data. Presynaptic terminals are assumed to be either highPr or low Pr and to contain only P/Q-type Ca2+ channels (QQ), only N-type (NN), or a mixture of the two (NQ) in the same relative proportions for both high and low Pr sites. When N-type channels are blocked by adding ω-CTx GVIA, NN-type terminals are blocked completely, QQ-type terminals are unaltered, andNQ-type terminals have theirPr either reduced or unaffected, depending on the initial Pr. A similar argument applies to the block of P/Q-type channels by ω-Aga. For further details, see Discussion. The percentages shownabove the control terminals are the estimated relative number of terminals in each category when the model was optimized to fit our data (Table 1). The pie graphs give the percentages of functional high and low Prterminals in each condition.
Fig. 4.
Fig. 4.
Summary of the progressive block experiments shown in Figures 1, 2, 3. Error bars represent mean ± SEM;stars indicate a statistically significant difference from control (one star, p < 0.05;two stars, p < 0.02). The progressive block time constants are increased by Cd2+ but are unaffected by the toxins (A, B). The percentage of high Pr terminals is unaffected by Cd2+ but is reduced by the toxins (C), implying that the toxins cause a population shift from high Pr to lowPr terminals.
Fig. 5.
Fig. 5.
Block of NMDA EPSCs by toxin is similar in control cells (i.e., with both high and low Prterminals contributing; A) or after 30 stimuli in 2 μm MK-801 (i.e., after most highPr terminals have been masked;B). This suggests that both highPr and low Prterminals contain, on average, the same mix of presynaptic Ca2+ channel subtypes. A, B, The above experiment was performed by using 1 μm ω-CTx GVIA (ω-CTx). Each panel was obtained from a different cell. A similar protocol was used for 0.5 μm ω-Aga. C, Summary of experiments of the type shown in A andB. Error bars represent mean ± SEM. The amount of block by each toxin is not significantly different in control or after 30 stimuli in 2 μm MK-801.
Fig. 1.
Fig. 1.
Progressive block of NMDA EPSCs by the use-dependent open channel blocker MK-801 can be used to estimate the probability of glutamate release, Pr.A, Averaged normalized NMDA EPSC amplitudes plotted against stimulus number for three different kinds of experiments, shown by different symbols. Each point is the ensemble average (± SEM) across different cells. Triangles, EPSC amplitude time course in normal bath solution without MK-801, showing the stability of the EPSCs over the duration of a typical experiment (n = 3 cells). Filled circles, EPSC amplitude time course in bath solution containing 2 μmMK-801, applied at stimulus 0 and maintained until the end of the recording (n = 9). Thesuperimposed solid line is a double exponential fit, suggesting the existence of at least two groups of terminals, one with a high Pr and the other with a lowPr. Open circles, EPSC amplitude time course after the removal of MK-801 at 30 stimuli, showing that the MK-801 block is irreversible under our conditions (n = 4). B, Left, Representative NMDA EPSCs recorded from one cell in control solution (trace labeled Con) and at 1,10, and 30 stimuli after 2 μm MK-801 has been added. Stimulus artifacts have been blanked. Right, The same EPSCs normalized at their peaks, showing that their decay is faster in the presence of MK-801 and does not change with stimulus number. This confirms that a homogeneous population of NMDA channels is being activated.
Fig. 2.
Fig. 2.
Control experiments confirm that MK-801 block measures Pr. A, Nonselective partial blockade of presynaptic Ca2+ currents by Cd2+ uniformly reduces Pr at both high and low Pr terminals.Left, Representative time course plot for one experiment, showing the block caused by Cd2+ (3.5 μm) and MK-801 (2 μm). Periods of drug application are indicated by horizontal bars. At the end of the experiment 100 μmd-APV was added, completely blocking the current and confirming that these were pure NMDA EPSCs. Right, Normalized progressive block plots averaged as in Figure 1A (n = 8 cells). The superimposed solid line is a double exponential fit with time constants shown in the inset; the dashed line is the control fit from Figure1A. Both block time constants are twice the corresponding control values (Fig. 1A), indicating a uniform halving of Pr by this concentration of Cd2+. B, Paired-pulse depression, which reflects Pr averaged across functioning terminals, is reduced after most highPr terminals have been masked by applying 30 stimuli in 2 μm MK-801. All traces are averages of 10 sweeps and were obtained from the same cell in drug-free external solution before (left) and after (right) the stimuli in MK-801. The interstimulus interval was 70 msec. Stimulus artifacts were not blanked.
Fig. 3.
Fig. 3.
Selective blockade of different Ca2+channel subtypes by ω-CTx GVIA (ω-CTx; A) orω-Aga (B) has little effect on progressive block time constants but reduces the proportion of highPr terminals. Left panels, Representative time course plots for individual experiments.Horizontal bars show the periods of application of ω-CTx GVIA (1 μm), ω-Aga (0.5 μm), MK-801 (2 μm), and d-APV (100 μm). The progressive block in B(left) is shown expanded in the inset.Right panels, Normalized progressive block plots averaged as in Figure 1A (n = 8 in A; n = 4 in B). The superimposed solid line in each panel is a double exponential fit with time constants shown in the inset; the dashed line is the control fit from Figure1A. The fitted time constants are similar to control, but the area under the fast component, which gives the proportion of high Pr terminals, is reduced.
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