Persistent accumulation of calcium/calmodulin-dependent protein kinase II in dendritic spines after induction of NMDA receptor-dependent chemical long-term potentiation

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.

Figure 3.

Persistent NMDA receptor-dependent accumulation of GFP-CaMKIIα in spines during cLTP. A, Projection image of a dendritic branch of a CA1 pyramidal neuron showing clearly identifiable spines. B, Images of a smaller dendritic region taken before and 60 min after application of cLTP-inducing protocol. Fluorescence intensity is represented in pseudocolor. Arrows indicate spines with an increase in ΔF/F. Scale bars: A, B, 2 μm. C, Images of a single spine before and after the induction procedure. Inset, Pseudocolor lookup table. D, Graph of F along the line indicated in C. E, Change of the fluorescence intensity (ΔF/F) over time for spine in C. Horizontal bar here and in F indicates drug application. F, Summary graph showing average temporal dynamics of fluorescence for all 135 of the spines with increasing ΔF/F in 11 experiments in which the LTP induction protocol was applied without APV (red) or in the presence of APV (7 spines in 5 experiments, blue). Black indicates summary graph for all 43 of the spines in three experiments in which no drugs were applied. G, Percentage of spines increasing ΔF/F for at least 60 min after drug application in the presence or absence of APV; asterisks indicate statistically significant data.

Figure 1.

Method for calculation of fluorescence values in spines and dendrites. A, B, Green-red overlay images of small dendritic region from a cell expressing GFP-CaMKIIα (green) and mRFP (red) before (A) and after (B) superimposition of a thresholding mask (blue). Masks for green and red channels were slightly different because they were constructed on the basis of background fluorescence for each channel. For simplicity, only the mask for the green channel is shown. The image is a projection of seven images in a stack with 0.5 μm z-steps. C, Average fluorescence of all of the pixels not covered by the mask within the dendritic ROI shown in B and measured for both the green (green line) and red (red line) channel along the z-axis. Background fluorescence was subtracted.

Figure 5.

Increase in spine CaMKII accumulation cannot be accounted for by an increase in spine volume. A, Expression of GFP-CaMKIIα (green) and mRFP (red) in the same dendritic branch. B, GFP-CaMKIIα (top) and mRFP (bottom) expression in a single spine before and 70 min after LTP induction, shown in pseudocolor. C, Change over time in s/d fluorescence ratio for GFP-CaMKIIα (green) and mRFP (red) for the spine shown in B. D, Summary graph for 18 spines showing an increased s/d ratio. The horizontal bar indicates cLTP induction. norm, Normalized. E, Summary graph from three control experiments without cLTP induction (29 spines). Scale bars: A, 2 μm; B, 1 μm.

Figure 2.

Induction of chemically induced, NMDA receptor-dependent LTP in slices transfected with GFP-CaMKIIα. A, GFP-CaMKIIα expression in hippocampal neurons in slice culture 13 d in vitro at day 4 after transfection. B, Zoomed image of the region indicated in A shows expression in all of the cellular compartments. Scale bar, 40 μm. C, Summary graphs of synaptic potentiation in control ACSF without APV (▪), in the presence APV, 100 μm (▵), or when no induction procedure was applied (•). The induction procedure is indicated by the horizontal bar. Each data point is an average of three responses. D, Average of five fEPSPs before (thin line) and 120 min after (thick line) induction. Calibration: 50 μV, 20 msec.

Figure 4.

In many spines, GFP-CaMKIIα is already accumulated under basal conditions. Distribution of s/d fluorescence intensity for red (gray bars) and green (open bars) channels in control conditions (n = 20) is shown. The average s/d fluorescence ratio for the green channel was significantly larger, 0.87 ± 0.06, than that for the red channel, 0.43 ± 0.03 (p < 0.01), indicating that bound GFP-CaMKIIα is already present in spines before the induction of cLTP. The calculated fraction of the GFP-CaMKIIα bound fraction in spines is 0.50 or 50% (see Materials and Methods).

Figure 6.

The fraction of the GFP-CaMKIIα bound pool in spines increases after induction of LTP. A, Dynamic of change in the fraction of bound CaMKIIα pool in spines. The bound fraction was calculated as described in Materials and Methods. The horizontal bar indicates the period of cLTP induction. B, The cLTP-induced change in the bound fraction of CaMKII is reversely correlated with the initial value before cLTP induction. Each filled circle represents the result of a single experiment.

Figure 7.

cLTP induction results in persistent accumulation of CaMKIIα at the PSD. A, B, Electron micrographs of hippocampal synapses labeled for CaMKIIα under control conditions (A) and 1 hr after induction of cLTP (B). Silver-enhanced gold particles appear as irregular black grains. Asterisks indicate grains counted as PSD-associated CaMKII labeling. Scale bar, 100 nm. C, Cumulative distribution of densities of gold label for CaMKIIα at individual PSDs from slice cultures under control conditions (▴) and after induction of chemical LTP (•).