Calcium threshold shift enables frequency-independent control of plasticity by an instructive signal

Abstract

At glutamatergic synapses, both long-term potentiation (LTP) and long-term depression (LTD) can be induced at the same synaptic activation frequency. Instructive signals determine whether LTP or LTD is induced, by modulating local calcium transients. Synapses maintain the ability to potentiate or depress over a wide frequency range, but it remains unknown how calcium-controlled plasticity operates when frequency variations alone cause differences in calcium amplitudes. We addressed this problem at cerebellar parallel fiber-Purkinje cell synapses, which can undergo LTD or LTP in response to 1-Hz and 100-Hz stimulation. We observed that high-frequency activation elicits larger spine calcium transients than low-frequency stimulation under all stimulus conditions, but, regardless of activation frequency, climbing fiber (CF) coactivation provides an instructive signal that further enhances calcium transients and promotes LTD. At both frequencies, buffering calcium prevents LTD induction and LTP results instead, identifying the enhanced calcium signal amplitude as the critical parameter contributed by the instructive CF signal. These observations show that it is not absolute calcium amplitudes that determine whether LTD or LTP is evoked but, instead, the LTD threshold slides, thus preserving the requirement for relatively larger calcium transients for LTD than for LTP induction at any given stimulus frequency. Cerebellar LTD depends on the activation of calcium/calmodulin-dependent kinase II (CaMKII). Using genetically modified (TT305/6VA and T305D) mice, we identified α-CaMKII inhibition upon autophosphorylation at Thr305/306 as a molecular event underlying the threshold shift. This mechanism enables frequency-independent plasticity control by the instructive CF signal based on relative, not absolute, calcium thresholds.

Keywords: Purkinje cell; calcium/calmodulin-dependent kinase II; cerebellum; long-term depression; long-term potentiation.

Fig. 1.

Spine calcium transients evoked by LTP- and LTD-inducing PF and CF activation patterns. (A–D) OGB-2 measurements. (A, Left) Purkinje cell filled with Alexa 633. (Scale bar: 20 μm.) (A, Center) Enlarged view. The circle outlines the ROI. (Scale bar: 1 μm.) (A, Right) Green fluorescence of OGB-2. The circle outlines the ROI. (Scale bar: 1 μm.) (B, Top) Electrophysiological responses to the following stimuli: 100-Hz PF burst + CF, PF burst alone, single-pulse PF + CF, and PF pulse alone. (Scale bars: vertical, 20 mV; horizontal, 100 ms.) (B, Bottom) Simultaneously recorded calcium transients. (Scale bars: vertical, 0.1 δG/R; horizontal, 500 ms.) (C) Calcium transients averaged from all Purkinje cell recordings (n = 11). Calcium signals are expressed as the percentage of the peak amplitude in each recording. (D) Bar graph summarizing calcium signal amplitudes (ΔG/R; average over a 200-ms period starting at stimulus onset, n = 11). (E–H) Fluo-5F measurements. (E, Left) Purkinje cell filled with Alexa 633. (Scale bar: 20 μm.) (E, Center) Enlarged view. The circle outlines the ROI. (Scale bar: 1 μm.) (E, Right) Green fluorescence of Fluo-5F. The circle outlines the ROI. (Scale bar: 1 μm.) (F) Electrophysiological responses and calcium transients arranged as in B. (Scale bars: Top, vertical, 20 mV; Top, horizontal, 100 ms; Bottom, vertical, 0.5 δG/R; Bottom, horizontal, 500 ms.) (G) Averaged calcium transients (n = 11). (H) Bar graph summarizing signal amplitudes (n = 11). Error bars indicate SEM. **P < 0.01; *P < 0.05.

Fig. S1.

Calcium transients evoked in spines and adjacent shaft areas by LTP- and LTD-inducing PF and CF activation patterns. (A–D) OGB-2 measurements. (A, Left) Purkinje cell filled with Alexa 633. (Scale bar: 20 μm.) (A, Center) Enlarged view. The circles outline the spine and shaft ROIs. (Scale bar: 1 μm.) (A, Right) Green fluorescence of OGB-2. The circles outline the spine and shaft ROIs. (Scale bar: 1 μm.) (B, Top) Electrophysiological responses to the following stimuli: 100-Hz PF burst + CF, PF burst alone, single-pulse PF + CF, PF pulse alone. (Scale bars: vertical, 20 mV; horizontal, 100 ms.) (B, Middle) Simultaneously recorded spine calcium transients. (B, Bottom) Shaft calcium transients. (Scale bars: vertical, 0.1 δG/R; horizontal, 500 ms.) (C) Bar graph summarizing calcium signal amplitudes in spines (ΔG/R; average over a 200-ms period starting at stimulus onset, n = 11). PF pulse alone: 0.02 ± 0.02 ΔG/R. PF + CF pulse: 0.06 ± 0.01 ΔG/R (P = 0.001; here and in the following, the P values refer to the statistical comparison with the previously stated stimulus condition/calcium signal amplitude); 100-Hz PF burst: 0.18 ± 0.02 ΔG/R (P = 0.00005); 100-Hz PF burst + CF: 0.24 ± 0.04 ΔG/R (P = 0.041). (D) Bar graph summarizing calcium signal amplitudes in shafts (n = 11). PF pulse alone: 0.02 ± 0.01 ΔG/R. PF + CF pulse: 0.03 ± 0.01 ΔG/R (P = 0.588); 100-Hz PF burst: 0.19 ± 0.03 ΔG/R (P = 0.0002); 100-Hz PF burst + CF: 0.24 ± 0.05 ΔG/R (P = 0.083). (E–H) Fluo-5F measurements. (E, Left) Purkinje cell filled with Alexa 633. (Scale bar: 20 μm.) (E, Center) Enlarged view. The circles outline the spine and shaft ROIs. (Scale bar: 1 μm.) (E, Right) Green fluorescence of Fluo-5F. The circles outline the spine and shaft ROIs. (Scale bar: 1 μm.) (F) Electrophysiological responses and spine/shaft calcium transients arranged as in B. (Scale bars: Top, vertical, 20 mV; Top, horizontal, 100 ms; Middle and Bottom, vertical, 0.5 δG/R; Middle and Bottom, horizontal, 500 ms.) (G) Bar graph summarizing calcium signal amplitudes in spines (n = 11). PF pulse alone: −0.01 ± 0.01 ΔG/R. PF + CF pulse: 0.04 ± 0.01 ΔG/R (P = 0.035); 100-Hz PF burst: 0.46 ± 0.15 ΔG/R (P = 0.013); 100-Hz PF burst + CF: 0.59 ± 0.19 ΔG/R (P = 0.039). (H) Bar graph summarizing calcium signal amplitudes in shafts (n = 11). PF pulse alone: 0.01 ± 0.004 ΔG/R. PF + CF pulse: 0.034 ± 0.012 ΔG/R (P = 0.07); 100-Hz PF burst: 0.36 ± 0.1 ΔG/R (P = 0.005); 100-Hz PF burst + CF: 0.46 ± 0.11 ΔG/R (P = 0.008). Values are shown as mean ± SEM. *P < 0.05; **P < 0.01.

Fig. S2.

Calcium transients monitored with the low-affinity calcium indicator OGB-5N. (A, Left) Purkinje cell filled with Alexa 633. (Scale bar: 20 μm.) (A, Right) Enlarged view. (Top) Green fluorescence of OGB-5N. The circle outlines the ROI. (Scale bar: 2 μm.) (Bottom) Corresponding Alexa 633 image. The circle outlines the ROI. (Scale bar: 2 μm.) (B) Electrophysiological responses to 100-Hz PF burst + CF stimulation (Left) and PF burst stimulation alone (Right). (C) Calcium transients recorded simultaneously with the electrical responses shown above. (D) Bar graph summarizing calcium signal amplitudes (ΔG/R; average over a 200-ms period starting at stimulus onset, n = 9). (E) Calcium transients averaged from all Purkinje cell recordings (n = 9). (Left) Calcium transients expressed as ΔG/R. (Right) ΔG/R values expressed as the percentage of the peak amplitude in each recording. Error bars indicate SEM. *P < 0.05.

Fig. 2.

In the low- and high-frequency activation range, BAPTA reverses LTD toward LTP. (A) Application of the 1-Hz LTD protocol results in LTP when BAPTA (20 mM) is added to the pipette saline. (Left) Time graph showing that LTD is induced under control conditions (n = 6), but LTP is induced in the presence of BAPTA (n = 6). (Center) Traces show EPSCs before and after tetanization. (Right) Individual cell data (t = 31–35 min). (B) Application of the 100-Hz LTD protocol results in LTP when BAPTA (5 mM) is added to the pipette saline. (Left) Time graph showing LTD under control conditions (n = 6) and LTP in the presence of BAPTA (n = 7). (Center) Typical traces. (Right) Individual cell data. Arrows indicate tetanization. Error bars indicate SEM. **P < 0.01.

Fig. S3.

BAPTA prevents LTD induction and reduces calcium transient amplitudes. (A) Application of the 1-Hz LTD protocol results in LTP when BAPTA (20 mM) is added to the pipette saline. (Left) Time graph showing that LTD is induced under control conditions (n = 6), but LTP is induced in the presence of BAPTA (n = 6). (Center Left) Traces show EPSCs before and after tetanization. (Center Right) Plot of individual cell data (t = 31–35 min). (Right Upper) Calcium transients (ΔG/R) evoked by single-pulse PF + CF activation (green trace), single-pulse PF activation (blue trace), and single-pulse PF + CF activation in the presence of BAPTA (purple trace). (Right Lower) Bar graphs show the averaged ΔG/R values determined for the low-frequency stimulus condition (Left) and the high-frequency stimulus condition (Right). (B) Application of the 100-Hz LTD protocol also results in LTP when BAPTA (5 mM) is added to the pipette saline. (Left) Time graph showing LTD under control conditions (n = 6) and LTP in the presence of BAPTA (n = 7). (Center Left) Typical traces. (Center Right) Plot of individual cell data (t = 31–35 min). (Right) Calcium transients (ΔG/R) evoked by 100-Hz PF burst + CF activation (red trace), PF burst activation (orange), and PF burst + CF activation in the presence of BAPTA (brown). Error bars indicate SEM. Note that in the bar graphs shown, all statistical comparisons between non-BAPTA conditions are done using the paired Student’s t test (because these recordings were obtained from the same cells), whereas comparisons between BAPTA and non-BAPTA conditions are done using the Mann–Whitney U test (recordings from different cells). *P < 0.05; **P < 0.01. Note that the panels showing electrophysiological data correspond to Fig. 2.

Fig. 3.

LTP induced by 100-Hz PF burst stimulation, but not 1-Hz PF stimulation, is prevented in mice that express CaMKII and cannot undergo Thr305/306 phosphorylation. (A, Left) Time graph showing that 1-Hz PF stimulation induces LTP in WT mice (n = 7) and TT305/6VA mice (n = 8). The arrow indicates time of tetanization. (A, Center) Typical traces show EPSCs before and after tetanization in WT mice (Top) and TT305/6VA mice (Bottom). (A, Right) Individual cell data (t = 36–40 min). (B, Left) Time graph showing that 100-Hz PF burst stimulation induces LTP in WT mice (n = 12), but LTD in TT305/6VA mice (n = 15). (B, Center) Typical traces show EPSCs before and after tetanization in WT (Top) and TT305/6VA mice (Bottom). (B, Right) Individual cell data. Error bars indicate SEM. **P < 0.01.

Fig. 4.

LTD is prevented in T305D mice, in which Thr305 replacement by Asp mimics constitutive inhibitory CaMKII autophosphorylation. (A, Left) Time graph showing that 1-Hz PF + CF activation induces LTD in WT mice (n = 8), but LTP in T305D mice (n = 6). (A, Center) Typical traces show EPSCs before and after application of the 1-Hz LTD protocol in WT mice (Top) and T305D mice (Bottom). (A, Right) Individual cell data (t = 36–40 min). (B, Left) Time graph showing that 100-Hz PF burst + CF activation induces LTD in WT mice (n = 6), but LTP in T305D mice (n = 7). (B, Center) Typical traces show EPSCs before and after application of the 100-Hz LTD protocol in WT mice (Top) and T305D mice (Bottom). (B, Right) Individual cell data. Error bars indicate SEM. **P < 0.01.

Fig. S4.

LTD is not impaired in TT305/6VA mice. (A, Left) Time graph showing that 1-Hz PF + CF activation induces LTD in WT mice (n = 8) and TT305/6VA mice (n = 5). (A, Center) Typical traces show EPSCs before and after application of the 1-Hz LTD protocol in WT mice (Top) and TT305/6VA mice (Bottom). (A, Right) Plot of individual cell data (t = 36–40 min). (B, Left) Time graph showing that 100-Hz PF burst stimulation paired with CF coactivation induces LTD in WT mice (n = 6) and TT305/6VA mice (n = 5). (B, Center) Typical traces show EPSCs before and after application of the 100-Hz LTD protocol in WT mice (Top) and TT305/6VA mice (Bottom). (B, Right) Plot of individual cell data (t = 36–40 min). Error bars indicate SEM.

Fig. S5.

LTP is not impaired in T305D mice. (A, Left) Time graph showing that 1-Hz PF activation induces LTP in both WT mice (n = 7) and T305D mice (n = 5). (A, Center) Typical traces show EPSCs before and after application of the 1-Hz LTP protocol in WT mice (Top) and T305D mice (Bottom). (A, Right) Plot of individual cell data (t = 36–40 min). (B, Left) Time graph showing that 100-Hz PF burst stimulation induces LTP in WT mice (n = 12) and T305D mice (n = 6). (B, Center) Typical traces show EPSCs before and after application of the 100-Hz LTP protocol in WT mice (Top) and T305D mice (Bottom). (B, Right) Plot of individual cell data (t = 36–40 min). Error bars indicate SEM.

Fig. 5.

Sliding plasticity thresholds and role of CaMKII inhibitory autophosphorylation. (A) Schematic presents a model of the relationship between calcium amplitudes and LTD/LTP as assessed in this study (the dashed lines indicate that possible LTP thresholds were not investigated). The numbers below show approximations of peak [Ca²⁺]_i values, which were calculated from ΔG/R measures recorded at each stimulus condition. Note that these peak values were determined from individual protocol-typical stimuli, and not from complete stimulus trains (Fig. S6). [Ca²⁺]_i values are not presented for the low-amplitude signals (N.D.), because no reliable measures above noise could be obtained with the low-affinity indicator Fluo-5F. (B) Diagram showing the role of CaMKII in LTD induction. CaMKII indirectly promotes LTD (dashed arrow) by negative regulation of phosphodiesterase 1 (PDE1), and the resulting facilitation of a cGMP/protein kinase G (PKG) cascade, which ultimately removes a blockade of LTD induction pathways by protein phosphatase 2A (PP2A) (also ref. 30). Inhibitory autophosphorylation of CaMKII at Thr305/306 may disable this negative regulation of PP2A.

Fig. S6.

Absence of calcium build-up during prolonged tetanization. (A) Calcium transients during application of the high-frequency LTD protocol (100-Hz PF burst, followed 120 ms after stimulus onset by a CF pulse; this stimulus pattern is applied at 1 Hz for 5 min). Fluorescence was monitored once per minute of tetanization, for a period of 2 s (2 stimuli). In addition, the fluorescence was measured immediately after completion of the tetanization protocol. The traces show the average calcium transients recorded from six Purkinje cells. The calcium signals are expressed as ΔG/R (percentage increase from the baseline preceding tetanization). Note that we selected the strongest tetanization protocol for a proof-of-principle demonstration that calcium levels do not significantly plateau in-between stimuli during the entire tetanization period. (B) Bar graph showing the peak amplitude levels for responses 1(red bars) and 2 (purple bars) during each 2-s sweep (200-ms average starting from stimulus onset) and the baseline calcium accumulation recorded before stimulus onset (average over a 200-ms baseline period; gray bars), as well as following the end of tetanization (end). The corresponding imaging periods are shown in A for the first 2 s of image acquisition. At no time point did the calcium accumulation reach significance compared with the 200-ms baseline period preceding the onset of the first stimulus (n = 6; P > 0.05). Values are shown as mean ± SEM.