Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials

Abstract

We compared the transient increase of Ca2+ in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca2+ transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca2+ transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 micro(m) for the soma, were of comparable amplitude. The Ca2+ transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca2+ transients, respectively. A comparable dependence of spinous Ca2+ transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca2+ transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca2+ transients could encode small time differences between pre- and postsynaptic activity.

Figure 1

Imaging of Ca²⁺ transients in single spines at high spatial and temporal resolution. (A) Optical detection of dendritic spines in neocortical neurons. Highlight projection through a stack of 20 frames of a layer 5 pyramidal neuron at low and high magnification imaged with a multiphoton laser scanning microscope. The neuron was filled via a somatic whole-cell recording patch pipette with the calcium indicator CG-1 (100 μM). Basal dendritic branches were selected (Left, box) and imaged at high magnification (Right). Horizontal line shows the position of the scan line in the xy plane used for collecting line scan images. (B) AP-evoked fluorescence transients in a spine head. Upper trace shows whole-cell voltage recording of a somatic AP. Resting membrane potential was −61 mV. The AP was elicited by a brief current injection into the soma (700 pA, 10 ms). Lower trace shows relative fluorescence changes ΔF/F (without averaging) evoked by a single AP. The (ΔF/F)_max was 1.06 and the decay time constant 182 ms, derived from the single exponential fit (gray line). (C) EPSP-evoked fluorescence transients in an active spine head. Upper trace shows an EPSP recorded in the soma (9.1 mV, peak amplitude). Same experiment as in B. Somatic EPSP amplitudes were constant during an experiment and ranged from 0.5–12 mV in different experiments. Lower trace shows fluorescence transient evoked by a single EPSP in a spine. Decay time constant was 143 ms and (ΔF/F)_max was 0.91. (D) AP-evoked Ca²⁺ transients were mediated by voltage-dependent channels. Calcium fluorescence transients were recorded in a spine before and after blocking voltage-dependent Na⁺ or Ca²⁺ channels. Addition of the Na⁺ channel blocker tetrodotoxin (1 μM) blocked AP initiation and reduced (ΔF/F)_max to 2.0 ± 10.1% (n = 3) of control when the same current as used to evoke APs was injected into the soma. Addition of Cd²⁺ (100 μM) reduced (ΔF/F)_max to 3.9 ± 5.9% (n = 3) of control. Waveform of somatic APs was unchanged (not shown). EPSP-evoked Ca²⁺ transients were mediated by NMDAR channels and were modified by AMPAR channels. Addition of NBQX (2 μM) reduced the somatic EPSPs to about 21% (n = 3; not shown) of control values. The (ΔF/F)_max was reduced to 76 ± 15% (n = 4) of control. Addition of AP-5 (100 μM) reduced (ΔF/F)_max to 2.9 ± 6.6% (n = 8), whereas the EPSPs were reduced by <5%. All blockers were added to the bath solution and perfused for ≥10 min.

Figure 2

Calcium transients in spine and dendritic shaft. (A) EPSP-evoked calcium fluorescence transients restricted to a single spine. The dendritic shaft and other spines near the active spine (arrow, lower right) were tested for fluorescence changes. In one active spine, but not in neighboring spines, a calcium fluorescence transient (shaded traces) was recorded after a single EPSP, whereas only a very small fluorescence increase was detected in the adjacent dendritic shaft. In contrast, back-propagating APs evoked fluorescence transients of similar amplitudes and decay times in all spines shown as well as in the dendritic shaft (solid traces). (B) Gradient of EPSP-evoked Ca²⁺ transient amplitude between spine head and dendritic shaft. Fluorescence changes were recorded simultaneously in shafts and active spines. Graph shows the amplitude of the fluorescence transient (ΔF/F)_max evoked by a single EPSP in the dendritic shaft plotted against (ΔF/F)_max in the spine (•, CG-1; ⋄, OG-2; error bars represent ± SD, same in C). Unity line represents the points of same fluorescence amplitudes in shaft and spine. Amplitude in the dendritic shaft was 20 ± 10% (n = 11) of the amplitude in the spine head. (C) Ca²⁺ transients evoked by back-propagating APs in dendritic shaft and spines. Graph shows (ΔF/F)_max evoked by a single AP in the dendritic shaft plotted against (ΔF/F)_max of a single AP-evoked transient in the spine. Amplitude of an AP-evoked transient in the dendritic shaft was 89 ± 36% (n = 11) of the amplitude in the spine head.

Figure 3

Summation of spinous Ca²⁺ transients depends on relative timing of back-propagating AP and EPSP. (A) EPSP preceded by AP (AP–EPSP) examined with CG-1 (100 μM). Upper trace shows whole-cell recording in current clamp mode of a single AP that was followed, 50 ms later, by a single EPSP. Resting membrane potential was −60 mV. Lower trace show the fluorescence transient evoked by this sequence in an active spine head. Fluorescence transient was fitted with a single exponential, yielding a (ΔF/F)_max of 1.47. (B) EPSP preceded by AP (AP–EPSP) examined with OG-2 (500 μM). Upper trace shows whole-cell recording of the sequence AP–EPSP and lower trace the fluorescence transient evoked. Resting membrane potential was −62 mV and (ΔF/F)_max was 1.12. (C) EPSP followed by AP (EPSP–AP) examined with CG-1. Upper trace shows whole-cell recording of a single EPSP that was followed, 50 ms later, by a single AP. Lower trace shows the fluorescence transient evoked by this sequence in an active spine head where (ΔF/F)_max was 1.54. (D) EPSP followed by AP (EPSP–AP) examined with OG-2. Upper trace shows whole-cell recording of the sequence EPSP–AP and the lower trace shows the fluorescence transient evoked by it where (ΔF/F)_max was 1.28. (E) Effect of relative timing on EPSP- and AP-evoked fluorescence transients using CG-1 as indicator. Graph shows (ΔF/F)_max evoked by the sequence EPSP–AP plotted against (ΔF/F)_max evoked by the sequence AP–EPSP. Records with failures (<25%) were excluded. Unity line represents points of same (ΔF/F)_max for the two sequences. In each spine examined the (ΔF/F)_max depended on the relative timing of EPSP and back-propagating AP. The AP–EPSP sequence evoked a (ΔF/F)_max of 1.44 ± 0.36 (n = 14) and the AP–EPSP sequence evoked a (ΔF/F)_max of 1.72 ± 0.39. Average ratio of the (ΔF/F)_max evoked by the sequence EPSP–AP to that evoked by AP–EPSP was 121 ± 13%. (F) Fluorescence transients measured with OG-2. Graph shows the differences of fluorescence transients evoked by several patterns of paired APs and EPSPs. •, (ΔF/F)_max values evoked by sequences where a single AP and a single EPSP were shifted by 50 ms (stimulation protocols shown in B and D). AP–EPSP-evoked (ΔF/F)_max was 0.89 ± 0.40 (n = 8) and that evoked by the sequence EPSP–AP was 1.21 ± 0.5. Average ratio of the (ΔF/F)_max values evoked by the sequence EPSP–AP to that evoked by the sequence AP–EPSP was 143 ± 32%. (ΔF/F)_max values evoked by a burst of two or three EPSPs (at 20 Hz) paired with two or three APs (at 20 Hz) but shifted by 15 ms had an average ratio of 122 ± 21% (○, two EPSPs and two APs, n = 3 and ■, three EPSPs and three APs, n = 3). In three of these experiments, AP-5 was subsequently added, reducing the average ratio to 92 ± 20%. Sequences of three EPSPs (at 20 Hz) followed or preceded by a single AP with a time interval of 15 ms (▵, n = 2) evoked very different (ΔF/F)_max values. Average ratio was 179 ± 39%.

Figure 4

Back-propagating APs reduce or increase Ca²⁺ inflow through NMDAR channels. (A) Calcium fluorescence transients evoked by single and paired APs and EPSPs. Fluorescence transients were measured with OG-2 (500 μM) as indicator. Transients evoked by a single EPSP (top trace) or a single AP (second trace from top) were fitted with a single exponential (thick lines). Shaded areas in the lower two traces show the result of the linear addition of the two fitted responses to single APs or EPSPs, which were shifted by 50 ms with respect to each other. Fluorescence transients evoked by the sequence AP–EPSP (second trace from bottom) or by the sequence AP–EPSP (bottom trace) were also fitted with a single exponential beginning at the peak of the response (thick line). The reduction in fluorescence change evoked by an EPSP in the sequence AP–EPSP compared with that evoked by a single EPSP alone is illustrated by the difference between the shaded area and the thick line (second trace from bottom). The increase in fluorescence change evoked by an AP in the sequence EPSP–AP compared with that evoked by a single AP alone is shown in the bottom trace. (B) Sublinear and supralinear summation of fluorescence transients. Same experiments as shown in Fig. 3F (•). Amplitude of the fluorescence transient evoked by a single EPSP was reduced to 72 ± 19% (n = 8) of control when preceded by an AP (AP–EPSP). Amplitude of the fluorescence transient evoked by an AP that was preceded by an EPSP (EPSP–AP) was increased to 157 ± 62% (n = 8) of control. It was reduced to 80 ± 17% (n = 5) if it was preceded by an AP (AP–AP).