Effect of transfection with ATP binding-impaired αCaMKII K42M mutant under different conditions.

<p><b>(A)</b> Example pairs of AMPAR EPSC traces at –65 mV from nontransfected cells and cells transfected with wild type or mGFP-αCaMKII K42M variants in control ACSF or in the presence of 100 μM DL-APV or 1 μM TTX. <b>(B)</b> Each dot compares the EPSC in untransfected and transfected cells. Overexpression of wild-type mGFP-αCaMKII has no effect on AMPAR EPSCs in normal media (top right). Overexpression of kinetically dead mGFP-αCaMKII K42M mutant leads to suppression of AMPAR EPSCs in normal media (top left), but effect is blocked by APV or TTX (bottom panels). Thick solid lines correspond to linear fits having the stated slope.</p

Images of transfected and nontransfected neurons 2 days after single-cell electroporation of organotypic cultured hippocampal slices.

<p><b>(A)</b> and <b>(B)</b> show multiple neurons in the CA1 region that were transfected with both mCherry (for facilitation of transfected cell localization) and mGFP-αCaMKII K42M variant. Stimulating electrode was positioned 100–200 μm from CA1 cell body layer. <b>(C)</b> and <b>(D)</b> show neighboring transfected and nontransfected neurons with two patch-pipettes (highlighted by yellow dashed lines) in the whole-cell configuration for simultaneous recordings of EPSCs from these cells. <b>(A)</b> and <b>(B)</b> show images of mCherry overlapped with DIC image (to show nontransfected cells and electrodes), and (<b>C)</b> and <b>(D)</b> show images of GFP overlapped with DIC image (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123718#sec002" target="_blank">Methods</a>). The images are black and white, but they are colored with red and green to highlight separate filter sets used for different images.</p

Summary and statistical significance of effects of different forms of CaMKII on EPSC (from data in Fig 2B).

<p>Data is presented as the average EPSC normalized to EPSC in untransfected cell. Error bars indicate S.E.M. (*—corresponds to p < 0.05, and **—to p < 0.005).</p

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

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

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

Dynamics of single cells during a small morph in the presence and absence of recurrent excitation.

<p>(<b>A</b>) Trace of a single gamma cycle of a representative cell of the CA3 memory in environments 1 (solid line) and 2 (dotted line). For both wall shapes, an action potential is released both in the absence of active recurrent collaterals (left plot) and in their presence (right plot). Time is represented by the horizontal axis. Gray area designates the window between the first spike in the population and the onset of global inhibition. Cell voltage and input currents are shown on the ordinate. (<b>B</b>) Trace of a representative cell of the activated CA3 memory in environment 1 (solid line) and environment 2 (dotted line). Pattern completion can be observed in the presence of recurrent collaterals (right plot), but not in their absence (left plot). (<b>C</b>) Trace of a representative cell not included in the memory (solid line) and with high excitation in the small morph (dotted line). Recurrent collaterals are not effective because the cell is not part of an active memory. There is no mechanism to avoid any action potential released by cells not included in a pattern.</p

The EC/DG/CA3 model.

<p>(<b>A</b>) In our model of the EC/DG/CA3 system, the excitatory granule cells of DG receive convergent input from EC (2700∶1) combined with a delayed feedback inhibition (delay: 3.3±0.4 msec) imposed by local interneurons. Excitatory cells in CA3 receive convergent input from both EC (2900∶1) and DG (∼50∶1) together with delayed feedback inhibition from local interneurons (delay: 3.3±0.4 msec) and recurrent excitatory input (delay: 1 msec). (<b>B</b>) Delayed feedback inhibition mediates internal competition that selects which cell fires in a given gamma cycle. Trace of three sample cells with different strength of excitatory feedforward currents. Time is represented at the horizontal axis. Gray area designates the window between the first spike and the onset of global inhibition. Cell voltage and input currents are shown on the ordinate. (<b>C</b>) Rate maps of sample EC neurons shown for both extreme shapes. (<b>D</b>) Action potentials (red dots) with overlaid trajectory (gray line) and equivalent rate maps of sample DG and CA3 cells.</p

Effect of grid cell realignment on the CA3 population response to morphing.

<p>Reproduction of the results of Colgin et al. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003641#pcbi.1003641-Colgin1" target="_blank">[21]</a> (left box) is compared to our experimental results (right box). In the condition in which the animals are trained in the same arena at the same location (red line), morphing induces a graded decrease in population correlation if compared to the initial state (red solid line) and a graded increase in correlation if compared to the final state (red dotted line). Under this condition, grid cells do not realign <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003641#pcbi.1003641-Leutgeb2" target="_blank">[19]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003641#pcbi.1003641-Fyhn1" target="_blank">[40]</a>. In the condition in which the animals are trained in different arenas or in the same arena but at different locations (black line), morphing induces a sharp and deep decrease in correlation if compared to the initial state (black solid line) and a graded increase in correlation if compared to the final state (black dotted line). Under this condition, grid cells realign <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003641#pcbi.1003641-Fyhn1" target="_blank">[40]</a>.</p

Recurrent collaterals explain the differences in the response of DG and CA3 to morphing observed experimentally [19].

<p>(<b>A</b>) Progressive change in the PV correlation of DG (dashed blue line) and CA3 (dashed orange line). (<b>B</b>) Average rate overlap and (<b>C</b>) mean spatial correlation of individual cells rate maps as a function of morphing as observed experimentally (top) and its equivalent analysis of simulated data with best model fit (bottom), including the response of CA3 without recurrent collaterals (light gray line). (<b>D</b>) PV autocorrelation for large distances (50 cm) measured experimentally (left) and with model best fit (right). (<b>E</b>) Mean number of place fields measured experimentally (left) and with model best fit (right).</p