Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons

Abstract

The size of the readily releasable pool (RRP) of vesicles is critically important for determining the size of postsynaptic currents generated in response to action potentials. However, discrepancies in RRP estimates exist among methods designed to measure RRP size. In glutamatergic hippocampal neurons, we found that hypertonic sucrose application yielded RRP size estimates approximately fivefold larger than values obtained with high-frequency action potential trains commonly assumed to deplete the RRP. This discrepancy was specific for glutamatergic neurons, because no difference was found between sucrose and train estimates of RRP size in GABAergic neurons. A small component of the difference in excitatory neurons was accounted for by postsynaptic receptor saturation. Train estimates of vesicle pool size obtained using more stimuli revealed that action potential-elicited EPSCs did not truly reach a steady state during shorter trains, and RRP estimates were closer to sucrose estimates made in the same neurons. This suggested that reluctant vesicles may contribute to the total available pool. Two additional lines of evidence supported this hypothesis. First, RRP estimates from strongly depolarizing hyperkalemic solutions closely matched those obtained with sucrose. Second, when Ca2+ influx was enhanced during trains, train estimates of pool size matched those obtained with sucrose. These data suggest that glutamatergic hippocampal neurons maintain a heterogeneous population of vesicles that can be differentially released with varying Ca2+ influx, thereby increasing the range of potential synaptic responses.

Figure 2.

Train estimates of RRP size match sucrose estimates in GABAergic neurons. A, Representative response to exogenous application of 0.5 m sucrose. B, A representative IPSC train (20 stimuli; 10 Hz) recorded from the same neuron as in A. Presynaptic stimulus transients have been blanked for clarity. C, Representative cumulative IPSC area (Cumul. Area) values from the same 20 stimuli, 10 Hz train. The dashed line represents a linear regression fit to data points after 1.5 s to estimate the cumulative area at time 0. D, Summary of the total RRP charge, as determined with trains (cumulative area at time 0) and sucrose applications performed in the same neurons (n = 10). Error bars represent SEM.

Figure 1.

Estimates of RRP size using sucrose application and action potential trains do not match in glutamatergic neurons. A, Representative response to exogenous application of 0.5 m sucrose. In all figures, the horizontal bar denotes the time of exposure to the secretagogue. B, Representative EPSC train (40 stimuli; 20 Hz) recorded from the same neuron as in A. Presynaptic stimulus transients have been blanked for clarity. C, Representative cumulative EPSC amplitude (Cumul. Amp.) values from a 40 stimuli, 20 Hz train. The dashed line represents a linear regression fit to data points after 1.5 s to estimate the cumulative amplitude at time 0. D, Representative cumulative EPSC area values from the same 40 stimuli, 20 Hz train as in C. The dashed line represents a linear regression fit to data points after 1.5 s to estimate the cumulative area at time 0. Inset, Summary of the total RRP charge, as determined with trains (cumulative area at time 0) and sucrose applications in the same neurons. ^*p < 0.002 (n = 8). Error bars represent SEM.

Figure 3.

Postsynaptic factors contribute only slightly to the discrepancy in RRP estimates. A, Representative EPSCs in the presence and absence of 25 μm GYKI 52466. B, Representative EPSCs in the presence of 0, 200, and 1000 μm kynurenate. A, B, Presynaptic stimulus transients have been blanked for clarity. C, Summary of train estimates of RRP size, expressed as percentages of sucrose estimates of RRP size performed in the same neurons, in control conditions and in the presence of 25 μm GYKI 52466 (GYKI) or kynurenate (kyn). ^*p < 0.03 compared with train estimates made in control conditions; in all conditions, train estimates were significantly different from sucrose estimates made in the same neurons (n = 5-13 in each condition). Error bars represent SEM.

Figure 4.

Cross-depletion experiments reveal that EPSC trains deplete only a subset of the sucrose-accessible pool. A, The areas of EPSCs (open bar) and sucrose responses (filled middle bar) were measured at 0.5 s after high-frequency stimulation (100 pulses at 20 Hz) and compared with the areas of original responses. ^*p < 0.001 (n = 9 for EPSCs and 8 for sucrose). In separate cells, the sucrose response 0.5 s after a 40 pulse, 20 Hz train (n = 10) showed ∼25% depression, consistent with RRP discrepancies in Figure 3. B, The difference in inhibition at various times after 100 stimuli at 20 Hz (n = 3 for each point). Recovery from inhibition was fit with a single exponential; τ = 1.09 s (EPSCs; open circles) and 1.28 s (sucrose; filled circles). Fits were constrained to a maximum of 100%. C, The areas of paired EPSCs (open circles) and sucrose responses (filled circles) were measured at various times after an initial pair of responses (n = 5 for each point). Recovery from inhibition was fit with a single exponential; τ = 9.94 s (EPSCs) and 7.85 s (sucrose). Fits were constrained to a maximum of 100%. Error bars represent SEM.

Figure 5.

RRP size estimates using strong depolarizations match sucrose estimates. A, Representative responses to hyperkalemic solution (100 mm K⁺) application in the presence of 200 μm kynurenate, shown in the presence and absence of 1 μm NBQX. Dashed lines indicate the portion of the response used to estimate RRP size, with the NBQX-insensitive component subtracted out in the analysis. B, Summary of train, sucrose, or strong depolarization (100 K⁺) estimates of RRP size, expressed as percentages of the sucrose estimates of RRP size performed in the same neurons (n = 13). Error bars represent SEM.

Figure 6.

Increasing 20 Hz trains to 100 stimuli suggests multiple components of EPSC depression and a greater estimate of RRP size. A, Peak EPSC amplitude values, measured in the presence of 200 μm kynurenate, during 100 stimuli, 20 Hz trains, normalized to the initial peak amplitude to illustrate the amplitude depression (n = 8). The normalized amplitudes were fit with a double-exponential decay; τ₁ = 122 ms; τ₂ = 773 ms. B, Representative cumulative EPSC area (Cumul. Area) values from a 100 stimuli, 20 Hz train. The dashed line represents a linear regression fit to time 0 from data points between 1.5 and 2 s, and the dotted line represents a linear regression fit to time 0 from data points between 4.5 and 5 s. C, Summary of the total RRP charge, as determined with 20 Hz trains of 40 or 100 stimuli (stim.), expressed as a percentage of the sucrose estimates of RRP size performed in the same neurons; ^*p < 0.001 compared with estimates derived from 40 stimuli (n = 8). Error bars represent SEM.

Figure 7.

Raising [Ca²⁺]_o and increasing action potential duration increase train estimates of RRP size. A, Summary of the total RRP charge, as determined with 40 stimuli, 20 Hz trains recorded in the presence of 0.75 mm Ca²⁺, 2 mm Ca²⁺, 4 mm Ca²⁺, or 4 mm Ca²⁺ plus 100 μm 4-AP, expressed as a percentage of the sucrose estimates of RRP size performed in the same neurons; ^*p < 0.002 and ^**p < 0.042 compared with sucrose estimates of RRP size (n = 5-14 in each condition). B, The areas of EPSCs were measured at various times after high-frequency stimulation in 0.75 mm Ca²⁺ (filled triangles), 2 mm Ca²⁺ (filled circles), and 4 mm Ca²⁺ (open circles) and compared with the areas of original responses (n = 4-5 for each point). Recovery from inhibition was fit with a double exponential; weighted τ = 3.49 s (0.75 mm Ca²⁺), 1.37 s (2 mm Ca²⁺), and 1.82 s (4 mm Ca²⁺). Fits were constrained to a maximum of 100%. Error bars represent SEM.