Nonlinear [Ca2+] signaling in dendrites and spines caused by activity-dependent depression of Ca2+ extrusion

Abstract

Spine Ca2+ triggers the induction of synaptic plasticity and other adaptive neuronal responses. The amplitude and time course of Ca2+ signals specify the activation of the signaling pathways that trigger different forms of plasticity such as long-term potentiation and depression. The shapes of Ca2+ signals are determined by the dynamics of Ca2+ sources, Ca2+ buffers, and Ca2+ extrusion mechanisms. Here we show in rat CA1 pyramidal neurons that plasma membrane Ca2+ pumps (PMCAs) and Na+/Ca2+ exchangers are the major Ca2+ extrusion pathways in spines and small dendrites. Surprisingly, we found that Ca2+ extrusion via PMCA and Na+/Ca2+ exchangers slows in an activity-dependent manner, mediated by intracellular Na+ and Ca2+ accumulations. This activity-dependent depression of Ca2+ extrusion is, in part, attributable to Ca2+-dependent inactivation of PMCAs. Ca2+ extrusion recovers from depression with a time constant of 0.5 s. Depression of Ca2+ extrusion provides a positive feedback loop, converting small differences in stimuli into large differences in Ca2+ concentration. Depression of Ca2+ extrusion produces Ca2+ concentration dynamics that depend on the history of neuronal activity and therefore likely modulates the induction of synaptic plasticity.

Figure 1.

Activity-dependent slowing of the decay of [Ca²⁺] transients. All panels are from the same experiment. A, Image of a small dendritic branch (red channel). The green line indicates the position of the line scan for rapid [Ca²⁺] imaging. B, A typical experiment (top, red channel; bottom, green channel). The stimulus was a single AP followed by a train of 25 APs at 50 Hz (average of 10 trials). The fluorescence transient in response to the AP train can be seen in the spine and dendrite (Dendr.) as an increase in fluorescence in the green channel. To minimize photodamage, the excitation light was shuttered during intervals irrelevant for the analysis (shutter closed). Bottom, corresponding membrane voltage trace. C, [Ca²⁺] transients (top; spine, red; dendrite, black; average of 34 trials) evoked by a single AP (bottom). The traces represent the ratio of green over red fluorescence (G/R) as a fraction of (G/R)_max (G/R at saturating [Ca²⁺]) (see Materials and Methods). Blue lines indicate monoexponential fits to determine τ_decay. Traces are offset vertically for display. D, [Ca²⁺] transients evoked by AP trains (top, 80 Hz; middle, 50 Hz; bottom, 20 Hz). Blue lines indicate monoexponential or double-exponential fits to determine τ_decay (see Materials and Methods). Traces are offset vertically for display. E, Overlay of the decay phases of peak scaled [Ca²⁺] transients evoked by a single AP (thick trace) and 80 Hz AP train (thin trace) in the spine. Blue lines indicate exponential fits. F, [Ca²⁺] transient decay time constants as a function of AP train frequency. G, Normalized [Ca²⁺] transient peak amplitudes as a function of AP train frequency.

Figure 2.

The slowing of the decay of [Ca²⁺] transients is Ca²⁺ dependent. A–C, Low-affinity Ca²⁺ indicator (Fluo-4FF). A, [Ca²⁺] transients in a spine (left, single AP; right, AP trains of 20, 50, and 80 Hz). Inset, Overlay of the decay of peak scaled single AP (gray) and 80 Hz AP train (black) evoked [Ca²⁺] transients. B, [Ca²⁺] τ_decay as a function AP frequency (n = 17 cells). C, [Ca²⁺] transient amplitudes, normalized to the [Ca²⁺] transient amplitude evoked by single APs (same data set as in B). D–F, High-affinity Ca²⁺ indicator (Fluo-4). D, [Ca²⁺] transients in a spine (left, single AP; right, AP trains of 20, 50, and 80 Hz). Inset, Overlay of the decay of peak scaled single AP (gray) and 80 Hz AP train (black) evoked [Ca²⁺] transients. E, [Ca²⁺] transient decay time constants (τ_decay) (n = 7 cells). F, [Ca²⁺] transient amplitudes, normalized to the [Ca²⁺] transient amplitude evoked by single APs (same data set as in E). G, Normalized decay time constants recorded with Fluo4-FF (solid lines/circles; same data as in B) and Fluo-4 (dashed lines/squares; same data as in E). Error bars indicate SEM.

Figure 3.

The activity-dependent slowing of the decay of [Ca²⁺] transients is attributable to depression of Ca²⁺ extrusion mechanisms. A, Comparison of measured [Ca²⁺] transients (black) and simulated [Ca²⁺] transients (gray). Simulations were based on the amplitude and decay time of the [Ca²⁺] transient evoked by a single AP. B, Measured [Ca²⁺] transient amplitudes versus expected [Ca²⁺] transient amplitudes, based on the amplitude and decay time of the [Ca²⁺] transient evoked by a single AP (Eq. 4). The black line shows equality. C, Same as B, except the calculation (Eq. 4) used the decay time of the [Ca²⁺] transient at the end of AP trains. The black line shows equality. D, Top, The decay phase of a [Ca²⁺] transient evoked by an 80 Hz AP train in a spine (baseline subtracted). Bottom, Corresponding Γ_i versus time. Dotted lines indicate amplitude levels as a percentage of peak [Ca²⁺]. E, Γ_i versus [Ca²⁺] level (percentage of peak [Ca²⁺]). To facilitate comparison of the rate constants between cells, instantaneous rate constants were averaged over [Ca²⁺] level windows of ±10% (thin lines, individual cells; circles, averages; n = 17).

Figure 4.

Time constant of the recovery from depression of Ca²⁺ extrusion. A, [Ca²⁺] transients in a spine evoked by a weak (pulse width, 2–5 ms; left) and a strong (pulse width, 8–15 ms; right) depolarization in voltage clamp. B, Amplitudes of [Ca²⁺] transients produced by single APs (n = 5 cells; open bars) and depolarizations (3–15 ms; n = 18 cells; filled bars). C, τ_decay of [Ca²⁺] evoked by APs (open bars; n = 17) and depolarizations (filled bars; n = 18) (same data set as in B). D, Protocol for measuring the recovery of depression of Ca²⁺ extrusion in a spine. E, Time course of recovery from depression of Ca²⁺ extrusion (t = 0; decay time constant of train evoked [Ca²⁺] transient; n = 9). F, Same as E, except normalized with respect to the baseline extrusion time constant. Error bars indicate SEM.

Figure 5.

Ca²⁺ extrusion is via PMCA and Na⁺/Ca²⁺ exchangers. A, Example of [Ca²⁺] transients evoked by a weak (left) and strong (right) depolarization in control conditions (black) and calmidazolium (gray). B, τ_decay in calmidazolium (Calm.) and control (Ctrl) (open circles, individual cells; filled circles, averages; n = 6). C, τ_decay in calmidazolium relative to control. D, Example of a [Ca²⁺] transient evoked by a weak (left) and strong (right) depolarization in control conditions (black) and with Li⁺ replacing Na⁺ (gray; scale bars as in A). E, [Ca²⁺] τ_decay in Li⁺ and control (Ctrl) (open circles, individual cells; filled circles, averages; n = 6). F, τ_decay in Li⁺ normalized to control. Error bars indicate SEM.

Figure 6.

Na⁺/Ca²⁺ exchangers and PMCAs are substrates for activity-dependent depression of extrusion. A, τ_decay after 50 Hz trains of APs (open bars) or weak depolarization (filled bars) normalized to single stimuli [AP, n = 17; depolarization (Depol.), n = 18; calmidazolium (Calm.), n = 6; Li⁺, n = 6; thapsigargin plus ryanodine (Thaps. + Ryanod.), n = 4; *statistically significant difference, unpaired t test; **statistically significant difference compared with internal control; NS, not significant compared with depolarization, unpaired t test]. B, [Ca²⁺] transients evoked by a single (left) and a 50 Hz (right) train of weak depolarizations in control conditions (black) and Li⁺ (gray). C, τ_decay of [Ca²⁺] transients (open circles, individual cells; filled circles, averages; n = 6). D, [Ca²⁺] transients evoked by a single (left) and a 50 Hz (right) train of weak depolarizations in control conditions (black) and in calmidazolium (gray; scale bars as in B). E, τ_decay of [Ca²⁺] transients (open circles, individual cells; filled circles, averages; n = 6). Error bars indicate SEM.

Figure 7.

Depression of Ca²⁺ extrusion and Ca²⁺ influx through NMDAR-timing-dependent plasticity of Ca²⁺ extrusion. A, Simultaneous [Ca²⁺] imaging and glutamate uncaging. The image of a spine is shown. The green line indicates the position of line scans performed for Ca²⁺ imaging. Caged glutamate was photolyzed next to the spine head (red line). B, Examples of [Ca²⁺] transients evoked by a strong depolarization alone (depol.; left) and glutamate uncaging (uncag.), followed by strong depolarization after 250 ms (right). C, τ_decay of [Ca²⁺] transients evoked by strong depolarization after glutamate uncaging (uncag.) compared with control (Ctrl.; open circles, individual cells; filled circles, averages; n = 15 spines, 5 cells). D, Averages and SEM (**statistically significant difference from unity). Dendr., Dendrite. E, [Ca²⁺] transients (red, spine; black, dendrite) and NMDAR currents (blue) evoked by single glutamate uncaging stimuli (left) and trains of six stimuli at 10 Hz (right) (n = 14 spines, 7 cells). F, Glutamate uncaging evoked Ca²⁺ accumulations produced by single stimuli compared with trains of stimuli. Ca²⁺ accumulations were quantified as the integral of [Ca²⁺] normalized to the integral of NMDAR currents (open circles, individual cells; filled circles, averages; n = 14 spines, 7 cells). G, Ca²⁺ accumulation produced by trains of uncaging stimuli normalized to single stimuli as a function of [Ca²⁺] transient amplitude of the single stimulus response (Δ[Ca²⁺]). Ca²⁺ accumulations were calculated as in F. H, Ca²⁺ accumulation produced by trains of uncaging stimuli normalized to single stimuli as a function of I_NMDA of the single response. Ca²⁺ accumulations were calculated as in F.