PKA-C but not PKA-R redistributes to spines upon activation. (A, B) Representative two-photon images of PKA-C-mEGFP co-expressed with PKA-RIIα or PKA-RIβ at rest, or in the presence of norepinephrine (NE) or forskolin and IBMX (F + I). mCherry (magenta) was co-expressed to reveal the neuronal morphology. (C–E) Quantification and comparison of the spine enrichment index at the resting state (C) and upon activation (D & E). As in panel C from left to right, n (spines/neurons) = 53/11, 34/7, 33/6, and 36/7.

Characterization of the inseparable R-C. (A) Schematic of wildtype PKA versus R-C. In both cases PKA-C was C-terminally tagged by mEGFP. (B–C) Representative images (B), quantifications of resting distribution (C), and the distribution change upon stimulation by forskolin and IBMX (D) of R-C compared to PKA-RIIα-mEGFP and co-expressed PKA-C-mEGFP/PKA-RIIα. RIIα and RIIα+C data are from Fig. 1C. n (spines/neurons) = 48/10.

PKA regulation of synaptic plasticity cannot be sustained by an inseparable PKA variant. (A–C) Representative image (A), time course (B), and the degree of potentiation (C) at the indicated timepoints in panel B of single-spine LTP experiments as triggered by focal glutamate uncaging at the marked spines (gray dot). In panel B, both stimulated spines (solid circles) and non-stimulated control spines (open circles) are shown. As in panel C from left to right, n (spines, each from a different neuron) = 8, 7, 17, 11, 9.

AMPA receptor-mediated synaptic transmission requires wildtype dissociable PKA. (A–C) Representative traces (red) normalized to the paired control (blue) (insets) and scatter plots of paired AMPA (A) and NMDA (B) receptor currents and AMPA/NMDA receptor current ratios (C) from neighboring untransfected CA1 neurons and those transfected with shRNA against PKA-C and the indicated shRNA-resistant rescue constructs. Statistical p values were obtained using a sign test (MATLAB). From left to right, n (neuron pairs) = 13, 11, 11, and 15.

Spine enrichment indexes and the movement of PKA-C into spines upon activation are not dependent on the protein expression level. (A–B) Averaged spine enrichment indexes within individual neurons of PKA-C co-expressed with RIIα (A) and RIβ (B), and their linear fit at rest or under the indicated stimulation.

PKA-C translocation can be driven by norepinephrine at low concentrations. (A, B) Representative two-photon images (A) and the collective trace of PKA-C-mEGFP co-expressed with PKA-RIIα at rest, or in the presence of 2 μM norepinephrine (NE) or forskolin and IBMX (F + I). DsRed Express (magenta) was co-expressed to reveal the neuronal morphology. n (spines/neurons) = 16/4.

PKA-C and PKA-RIα differentially re-distributed upon activation. The collective trace of PKA-C-mEGFP co-expressed with PKA-RIα (orange) and PKA-RIα-mEGFP (light blue) at rest, or in the presence of 10 μM norepinephrine (NE) or forskolin and IBMX (F + I). DsRed Express was co-expressed to reveal the neuronal morphology. n (spines/neurons) = 20/5 for PKA-C and 16/4 for RIα.