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, 105 (33), 12039-44

Optical Induction of Plasticity at Single Synapses Reveals Input-Specific Accumulation of alphaCaMKII

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Optical Induction of Plasticity at Single Synapses Reveals Input-Specific Accumulation of alphaCaMKII

Yan-Ping Zhang et al. Proc Natl Acad Sci U S A.

Abstract

Long-term potentiation (LTP), a form of synaptic plasticity, is a primary experimental model for understanding learning and memory formation. Here, we use light-activated channelrhodopsin-2 (ChR2) as a tool to study the molecular events that occur in dendritic spines of CA1 pyramidal cells during LTP induction. Two-photon uncaging of MNI-glutamate allowed us to selectively activate excitatory synapses on optically identified spines while ChR2 provided independent control of postsynaptic depolarization by blue light. Pairing of these optical stimuli induced lasting increase of spine volume and triggered translocation of alphaCaMKII to the stimulated spines. No changes in alphaCaMKII concentration or cytoplasmic volume were observed in neighboring spines on the same dendrite, providing evidence that alphaCaMKII accumulation at postsynaptic sites is a synapse-specific memory trace of coincident activity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LTP induction by pairing of single action potentials in CA3 cells with ChR2-mediated depolarization of synaptically connected CA1 cells. (a) CA1 pyramidal cell in organotypic slice culture (maximum intensity projection) expressing ChR2, RFP, and Dronpa-α CaMKII. Arrows indicate SV40 polyA sequences. (b) Paired recording configuration to assess light-induced plasticity in transfected CA1 pyramidal cell. Blue spot indicates illuminated area. (c) LTP induction protocol. A single AP evoked by current injection (2 nA, 5 ms) into a patch-clamped CA3 pyramidal cell was paired with a short burst of 5 APs elicited by blue light illumination (200 ms) of a transfected CA1 pyramidal cell (Δt = 6 ms). Pairing was repeated 20 times at 0.1 Hz. (d) Light-induced bursting in postsynaptic cell (blue bar: 200 ms light pulses, 20 repetitions at 0.1 Hz) did not significantly change EPSC amplitudes in transfected CA1 pyramidal cells (107% ± 16%, n = 7 pairs). Pairing stimulation as described in c induced 274% ± 76% potentiation (n = 7 pairs). Insert: example traces before and after light stimulation. Error bars indicate SEM.
Fig. 2.
Fig. 2.
Spine volume changes induced by pairing of glutamate uncaging and ChR2 activation. (a) Setup for simultaneous uncaging, imaging, and ChR2 stimulation. Two Ti:Sapph lasers were regulated by electro-optical modulators (EOM) and coupled through a polarizing beamsplitter cube (POL). A high-power blue LED coupled in through the epifluorescence port excited ChR2. Dichroic mirrors were 700DCXXR (1, 2), 560DCXR (3), and Q505LP (4). Photomultiplier tubes (PMT) were protected by a shutter (S) during the blue light pulses. (b) To induce plasticity at individual synapses, single uncaging pulses were paired with 200 ms blue light pulses 20 times at 0.1 Hz (Δt = 0). uEPSCs are averages of five consecutive traces each. (c) Spine volume change induced by optical pairing protocol in the presence of MNI-glutamate (RFP signal, maximum projections). Time course shows transient and permanent component (black circles, n = 16 spines, 8 cells). No volume changes were induced in the absence of MNI-glutamate (gray triangles, n = 12 spines, 5 cells).
Fig. 3.
Fig. 3.
Enrichment of αCaMKII in spines. (a) CA1 pyramidal cell dendrite expressing GFP-αCaMKII and RFP (maximum intensity projections, grayscale saturated to show dim spines). Left: GFP-αCaMKII signal. Middle: Cytoplasmic volume (RFP). Right: Color-coded ratio of αCaMKII/volume (G/R), indicating elevated αCaMKII concentration in spines. (b) Cumulative distribution of G/R ratio (protein concentration) in spines relative to parent dendrite (S/D ratio). αCaMKII, but not GFP, is enriched in dendritic spines. GFP, n = 286 spines, 3 cells, median S/D ratio = 1.01; GFP-αCaMKII, n = 80 spines, 4 cells, median S/D ratio = 1.32. (c) FRAP analysis on a dendritic spine expressing YFP-αCaMKII and RFP. Black dots: YFP-αCaMKII fluorescence intensity in spine. Black line: single exponential fit. Note ≈20% unrecoverable fraction. Red dots: RFP fluorescence. Note rapid (insert) and complete recovery of fluorescence. RFP showed much faster recovery following photobleaching than YFP-αCaMKII (τ RFP = 0.31 s, τYFP-αCaMKII = 16 s). (d) Correlation between FRAP and spine/dendrite (s/d) ratio measurements in individual spines. YFP-αCaMKII TT305/306VA mutant showed higher unrecoverable fraction (fu) and higher enrichment in spines compared with wild-type αCaMKII. Black line: predicted correlation fu = 1 − 1/(s/d).
Fig. 4.
Fig. 4.
Input-specific accumulation of αCaMKII. (a) Maps of αCaMKII concentration. Stimulated spines (red dot) showed either transient (left) or persistent increase in volume and αCaMKII concentration (right). (b) Spine volume changes in response to optical pairing protocol. Stimulated spines were sorted into two groups according to their volume 30–40 min after pairing (transient spines: <30% change, n = 13; persistent spines, >30% change, n = 10). Right panel: neighboring, nonstimulated spines on the same dendrite show no significant volume change (mean distance from stimulated spine: 5.5 μm ± 0.9 μm, n = 110). (c) αCaMKII enrichment in spines: αCaMKII concentration (G/R) in spine normalized by αCaMKII concentration (G/R) in dendrite. Spines were sorted according to persistence of volume change as described in b. Significant αCaMKII enrichment was detected in persistently enlarged spines at t = 13 min (paired t test, one-tailed, P < 0.05), but not in the group that responded only with transient swelling. Note identical αCaMKII concentration in both groups before stimulation. Right panel: neighboring, nonstimulated spines showed no significant change in αCaMKII concentration. (d) Soluble and bound fraction calculated from data shown in c and b (see SI Text). In the spines that responded with persistent volume change to paired stimulation (middle panel), the amount of both bound and soluble αCaMKII was persistently increased by a factor of 2.0 and 1.6, respectively.

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