Integration of asynchronously released quanta prolongs the postsynaptic spike window

Abstract

Classically, the release of glutamate in response to a presynaptic action potential causes a brief increase in postsynaptic excitability. Previous reports indicate that at some central synapses, a single action potential can elicit multiple, asynchronous release events. This raises the possibility that the temporal dynamics of neurotransmitter release may determine the duration of altered postsynaptic excitability. In response to physiological challenges, the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus of the hypothalamus (PVN) exhibit robust and prolonged increases in neuronal activity. Although the postsynaptic conductances that may facilitate this form of activity have been investigated thoroughly, the role of presynaptic release has been largely overlooked. Because the specific patterns of activity generated by MNCs require the activation of excitatory synaptic inputs, we sought to characterize the release dynamics at these synapses and determine whether they contribute to prolonged excitability in these cells. We obtained whole-cell recordings from MNCs in brain slices of postnatal day 21-44 rats. Stimulation of glutamatergic inputs elicited large and prolonged postsynaptic events that resulted from the summation of multiple, asynchronously released quanta. Asynchronous release was selectively inhibited by the slow calcium buffer EGTA-AM and potentiated by brief high-frequency stimulus trains. These trains caused a prolonged increase in postsynaptic spike activity that could also be eliminated by EGTA-AM. Our results demonstrate that glutamatergic terminals in PVN exhibit asynchronous release, which is important in generating large postsynaptic depolarizations and prolonged spiking in response to brief, high-frequency bursts of presynaptic activity.

Figure 1.

Characterization of asynchronous glutamate release onto MNCs. A, Top, Evoked EPSPs (eEPSPs) exhibit a prolonged decay compared with sEPSP. Individual events are shown in gray (30 eEPSPs; 147 sEPSPs) and average events in black. Bottom left, Overlaid average traces of eEPSPs (black) and sEPSPs (gray). Bottom right, Peak scaled average spontaneous (gray) and evoked (black) EPSPs. B, In voltage-clamp mode, synaptic stimulation elicits EPSCs that are asynchronous. Top, Thirty trials at 0.2 Hz overlaid; bottom, same cell, 10 traces distributed. Stimulus artifacts have been removed for clarity. C, The number of asynchronous events after single stimuli decay exponentially with a time constant of 10.9 ms (n = 23).

Figure 2.

Asynchronous release is not polysynaptic or age dependent. A, Data from a single cell demonstrates that both the synchronous and asynchronous components of release have a similar threshold for activation. B, Representative traces obtained from a young (P22, black traces) and older (P43, orange traces) rat show that asynchronous release is evident at both ages. C, Correlation of charge transfer for the synchronous component versus the asynchronous component for different cells obtained from both young (P21–P28; n = 29; black) and older (P43–P44; n = 12; orange) animals. The R² values for the linear regression in C are 0.18 and 0.35 for the young and older animals, respectively.

Figure 3.

Asynchronous release does not depend on the phenotype of the postsynaptic cell. A, Whole-cell recordings were obtained from either GFP-positive (VP) or -negative (OT) neurons. B, GFP-positive and -negative cells had similar input resistances and responses to depolarizing current steps. C, Representative traces show no difference in the amount of asynchronous release between the two cell types.

Figure 4.

Asynchronous release is calcium dependent. A, Voltage-clamp traces from a single neuron show the effect of the membrane permeable calcium chelator EGTA-AM (25 μm, 15 min) on glutamate release. B, The summary graph from five cells demonstrates that there is a preferential decrease of the asynchronous release component. C, Increasing the concentration of EGTA-AM (100 μm) decreases asynchronous release to an even greater extent (n = 6). D, Continuous whole-cell recording with no pharmacological manipulations shows no rundown of asynchronous release over the 30 min.

Figure 5.

Buffering postsynaptic Ca²⁺ does not inhibit asynchronous release. A, Whole-cell recordings were first made with normal internal pipette solution. Evoked currents were recorded for a control period and the pipette was quickly withdrawn from the cell. The same cell was then repatched with a pipette containing 10 mm EGTA (n = 6). Evoked currents were recorded for up to 30 min with the new internal solution. B, There was no difference in evoked currents recorded with either normal (black traces) or EGTA containing pipette solution (gray traces, p > 0.05). Evoked currents used for analysis in B were obtained 16.7 ± 1.7 min after repatching the cell.

Figure 6.

Asynchronous release accumulates during repetitive afferent activity. A, B, Representative voltage-clamp traces during short (four pulse) trains at 20 (A) and 50 Hz (B). Top traces are control and bottom traces are after application of 25 μm EGTA-AM. Stimulation artifacts have been removed for clarity. C, Quantification of these data show that asynchronous charge transfer elicited by 20 and 50 Hz trains in control was 2.53 ± 0.44 pC and 2.79 ± 0.47 pC, respectively. After application of 25 μm EGTA-AM, asynchronous release was 1.28 ± 0.21 pC and 1.39 ± 0.20 pC for the 20 and 50 Hz trains respectively (*p < 0.05; n = 5). D, The synchronous component of release during the trains was not inhibited after EGTA-AM (20 Hz control, 2.23 ± 0.58 pC; 20 Hz EGTA-AM, 2.01 ± 0.60 pC; p > 0.05; 50 Hz control, 2.28 ± 0.52 pC; EGTA-AM, 1.94 ± 0.61 pC; p > 0.05; n = 5).

Figure 7.

Asynchronous release promotes prolonged spiking. A, B, Representative current-clamp traces during short (four pulse) trains at 20 Hz (A, top) and 50 Hz (B, top, 30 overlaid traces in each condition). Average spike histograms from eight cells are shown under each trace. C, Current-clamp traces at 50 Hz before (black) and after (orange) 25 μm EGTA-AM. D, Histogram plotting the average number of spikes/5 ms bin over time for the 50 Hz train, both in control (black) and after EGTA-AM (orange). The data represent the average from eight cells (30 trials per cell). E, The number of synchronous spikes evoked during the stimulation train both in control and after EGTA-AM (control vs EGTA, ANOVA, p > 0.05; n = 8). F, The number of spikes evoked in the 500 ms after the end of the stimulation train in control and after application of 25 μm EGTA-AM (control vs EGTA-AM, ANOVA, *p < 0.05; n = 8).