Effective release rates at single rat Schaffer collateral-CA1 synapses during sustained theta-burst activity revealed by optical imaging

Abstract

To understand how information is coded at single hippocampal synapses during high-frequency activity, we imaged NMDA receptor-mediated Ca(2+) responses in spines of CA1 neurons using two-photon microscopy. Although discrete quantal events were not readily apparent during continuous theta-burst stimulation (TBS), we found that the steady-state dendritic Ca(2+) response was spatially restricted (half-width < 1 microm), voltage dependent and sensitive to MK-801, indicating that that it was mediated by activation of NMDA receptors at single synapses. Partial antagonism of NMDA receptors caused a similar reduction of NMDA EPSCs (measured at the soma) and local dendritic Ca(2+) signals, suggesting that, like EPSCs, the steady-state Ca(2+) signal was made up of a linear addition of quantal events. Statistical analyses of the steady-response suggested that the quantal size did not change dramatically during TBS. Deconvolution of TBS-evoked Ca(2+) responses revealed a heterogeneous population of synapses differing in their capacity to signal high-frequency information, with an average effective steady-state release rate of approximately 2.6 vesicles synapse(-1)s(-1). To assess how the optically determined release rates compare with population measures we analysed the rate of decay of peak EPSCs during train stimulation. From these studies, we estimated a unitary vesicular replenishment rate of 0.02 s(-1), which corresponds to an average release rate of approximately 0.8-2 vesicles s(-1) at individual synapses. Additionally, extracellular recordings from single Schaffer collaterals revealed that spikes propagate reliably during TBS. Hence, during high-frequency activity, Schaffer collaterals conduct spikes with high fidelity, but release quanta with relatively lower efficiency, leaving NMDA receptor function largely intact and synapse specific. Heterogeneity in release rates between synapses suggests that similar patterns of presynaptic action potentials could trigger different forms of plasticity at individual synapses.

Figure 5. Release rate are heterogeneous between CA3–A1 synapses

Aa, exmples of TBS-evoked Ca²⁺ responses at different spines, normalized to the quantal response measured during LFS (scale bar 2 quanta). b, the instantaneous release rates estimated for each synapse shown in a are plotted alongside the traces. B, a histogram of the mean steady-state release rates obtained from 16 synapses (15 cells).

Figure 6. TBS-evoked AMPA and NMDA receptor-mediated EPSCs

A, AMPA EPSCs evoked by 60 s of TBS. Average of first (black) and last (grey) five responses of the train (bottom left), are normalized to the first EPSC in the burst (bottom right). B, the average amplitude of each burst is plotted as a function of time (n= 8 cells). The fraction of the response that recovers and subsequently undergoes depression during the train (predicted by eqn (S3); see Supplemental information), is indicated by the dashed grey line. The total recycling pool (TRP) is shaded in grey. C, NMDA EPSCs evoked by TBS. D, the average peak amplitude of AMPA and NMDA receptor components of EPSCs is plotted (n= 6 cells). E, the EPSC peak amplitude (average of each burst) in response to 6 consecutive trains (60 s TBS) applied at 5 min intervals.

Figure 1. Single spine Ca²⁺ responses to LFS and TBS

A, an image of a spine and dendrite. B, the relative intensity (ΔF/F₀) of the responses in the spine during: (a) LFS (superimposed grey traces are single trials and the black trace is the average quantal response, 0.1 Hz) and (b) continuous TBS (4 pulses at 50 Hz given every 200 ms). The vertical arrow in Ba indicates the time of stimulation. The steady-state amplitude of the TBS-evoked Ca²⁺ responses is indicated by the horizontal dotted line. C, the ΔF/F₀ along the vertical grey line in A is plotted as a function of space and time during continuous TBS.

Figure 7. High-fidelity conduction at single axons in the stratum radiatum

Aa, extracellular recordings from single axons show a characteristic ‘all-or-none’ response to increasing stimulus intensity. Ab, the peak spike amplitude is plotted against stimulus intensity. Bi, the response of the same axon as in A, to prolonged TBS (bursts of 4 pulses at 50 Hz delivered every 200 ms). Bb, the peak spike amplitude is plotted against stimulus number. C, binarized responses (0 failures, 1 success) from 6 axons were averaged to compute the probability of spike conduction (P_spike).

Figure 2. Ca²⁺ responses at steady-state are mediated by NMDA receptors and are spatially restricted

A, Ca²⁺ response from a spine in the absence (black trace) and presence of 10 μm MK-801 (grey trace). B, Ca²⁺ responses measured at +10 mV (black trace) and during a voltage ramp to −80 mV (grey trace). C, spatio-temporal Ca²⁺ profile along the dendrite during continuous TBS (diagonal arrow 10 s). D, the average spatial Ca²⁺ profile during the steady-state response was estimated by fitting it to a Gaussian function (black line).

Figure 3. Dendritic Ca²⁺ signals are linearly related to NMDA EPSC

A, the simultaneous measurement of NMDA receptor-mediated Ca²⁺ response in a single spine (illustrated on the right; the region of interest is depicted by the dashed black box 0.9 μm in width) (a) and somatic NMDA receptor-mediated EPSCs (V_hold=−20 mV) evoked by TBS (b). A raster of the instantaneous release rate estimated by deconvolving the TBS-evoked response with the fitted quantal response (peak amplitude 50%ΔF/F; inset; also see Fig. 4 and results for details). Ba, Ca²⁺ responses and, b, EPSCs evoked by the same stimulus, in the presence of subsaturating NMDA receptor antagonist AP5. The raster of the instantaneous release rate was estimated by deconvolving the TBS-evoked response with a quantum of similar kinetics, but with a peak amplitude of 26%ΔF/F (the height of the raster is also reduced to half the amplitude highlight this reduction). The steady-state release rate is noted besides the raster plots in A and B. The grey trace in Aa and Ba is the model train constructed by adding quanta according to the estimated release rate. The integral of the peak NMDA EPSC (C) and Ca²⁺ during the course of TBS (D). E, a plot of the relative integrated EPSC versus integrated Ca²⁺ signals during graded levels of AP5 block of NMDARs. Integrals were normalized to values in control conditions. Different levels of antagonism were achieved by varying the concentration of AP5 (1–5 μm; n= 6 cells).

Figure 4. Release rate estimated by deconvolution analysis

Aa, the TBS-evoked Ca²⁺ response at a single synapse (from Fig. 2C) decays to a steady state indicated by the horizontal dashed line. The average quantal response evoked during LFS is shown on the same scale as a, or at 5× (right; insets). The dashed grey line represents a double exponential fit: A (e^{(−x/τrise) −}e^{(−x/τdecay)}) of the quantal response. Ab, raster of the instantaneous release rates obtained by deconvolving the TBS-evoked response (Aa) with the fitted quantal response (Aa inset). Ac, a model train (grey trace), constructed by summing the quantal responses according to Ab, is compared with the TBS-evoked Ca²⁺ response. Ad, 1 Hz (light grey), 3 Hz (black) and 5 Hz (grey) Poisson trains, simulated with the average quantal response are illustrated. The horizontal line marks the mean of the 3 Hz train. B, the quantal size estimate (QSE) obtained during LFS, or from the mean-to-variance ratio of the steady-state responses during the real and simulated trains.