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

Author Posting. © Society for Neuroscience, 2004. This article is posted here by permission of Society for Neuroscience for personal use, not for redistribution. The definitive version was published in Journal of Neuroscience 24 (2004): 9324-9331, doi:10.1523/JNEUROSCI.2350-04.2004.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)-CaMKIIα 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-CaMKIIα is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIα in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIα in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIα. These results are consistent with the hypothesis that CaMKIIα accumulation at synapses is a memory trace of past synaptic activity.This work was supported by Grant R01 NS-27337 from the National Institutes of Health/National Institute of Neurological Disorders and Stroke

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

<div><p>Because CaMKII is the critical Ca²⁺ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca²⁺ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII <i>in vitro</i>. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.</p></div

Fast decay of Camui signal is not affected by Calyculin A, an inhibitor of PP1/PP2A.

<p>(A) Top panel: lifetime images before, during, and after uncaging showing that the lifetime change of Camui (Camui, LT) is restricted to the stimulated spine, as indicated by the change of pseudo-color from orange to yellow; the location of glutamate uncaging is indicated by an asterisk. Middle panel: Camui content of spines, as measured by single-photon counting of GFP fluorescence (Camui, SPC), dramatically increased in stimulated spine. Bottom panel: fluorescent images of the volume marker mCherry (Cherry, F) showing spine enlargement after glutamate uncaging. Scale bar units: top–ns/pixel, middle–photons/pixel, bottom–AU. (B) Fluorescence lifetime response of WT Camui produced by glutamate uncaging (average of 35 spine experiments, filled black circles) overlapped with fitted double exponential (green) and underlying the first (dash red) and the second (dash blue) exponentials; dendritic response–black squires. (C–E) Graphs showing effects of Calyculin A (open symbols) on WT Camui fluorescence lifetime (raw, C and scaled, D) and spine size (E) in comparison to control conditions (filled symbols). Glutamate uncaging protocol (eight pulses at 0.5 Hz) was started at time 0 (horizontal black bar).</p

T286D/T305A/T306A Camui mutant is further activated by spine stimulation and has deactivation similar to that of WT Camui.

<p>(A, B, and D) Graphs of fluorescent lifetime change after glutamate uncaging of WT Camui (filled symbols), T286D/T305A/T306A and T305A/T306A Camui mutants (open symbols), T286D/T305D/T306D—gray symbols; (A) and (D), raw and (B), scaled data. (C) Change in spine size. Glutamate uncaging protocol (eight pulses at 0.5 Hz, horizontal black bar) started at time 0. (E) Bar diagram of basal fluorescence lifetime change in different experimental conditions in comparison to basal lifetime of WT Camui. Shadow line at the bottom indicates SE of basal lifetime for WT Camui. Stars indicate statistical significance change relative the basal lifetime of WT Camui.</p