Elevated PSD-95 Blocks Ion-flux Independent LTD: A Potential New Role for PSD-95 in Synaptic Plasticity

Abstract

We recently demonstrated that NMDA receptors (NMDARs) are capable of ion-flux independent signaling through conformational change in the NMDAR intracellular domain resulting in long-term depression of synaptic transmission (LTD). Here we show that PSD-95 overexpression blocks agonist induced conformational movement in the NMDAR intracellular domain as well as LTD that is NMDAR-dependent and ion-flux independent. Interestingly, previous studies indicate that overexpressed PSD-95 does not block NMDAR-dependent LTD. These data support a model where ion-flux independent LTD is predominant in young animals, which have synapses with low amounts of PSD-95, whereas only ion flux dependent LTD occurs at more mature synapses, which have more PSD-95 that would block ion-flux independent LTD. These results may reconcile different findings regarding ion-flux independent LTD.

Keywords: MAGUK; NMDA receptor; conformational movement; long-term depression; metabotropic signaling; post-synaptic density protein 95.

Figure 1:. PSD-95 overexpression blocks NMDA-induced conformational movement in the NMDAR c-terminal domain

A) Experimental design. B) Field of neurons transfected with GluN1-GFP and infected with Sindbis expressing nuclear localization signal tagged (NLS)-mCherry. Scale bar, 20μm C) (Top) Representative images of neurons expressing GluN1-GFP/GluN1-mCherry and NLS-mCherry or NLS-mCherry + PSD-95 in a solution containing 100μM 7CK, (Bottom) same neurons in the presence of 100μM 7CK and 25μM NMDA. Pseudo-colored pixels indicating fluorescence lifetime of GluN1-GFP (see color scale); scale bar, 5μm. D) Average FRET efficiency for the indicated conditions; N>17 neurons per condition, individual data points for each neuron are superimposed; *p<0.05, paired t-test.

Figure 2:. PSD-95 overexpression blocks ion-flux independent LTD

A) Representative EPSCs obtained at −60 or +40mV in normal ACSF (black) or in the presence of 10μM L689 (gray). Shaded areas are analyzed in B. Scale bars, 10ms, 20pA throughout. B) Effect of L689 on EPSCs recorded at −60 or +40mV at peak amplitude (10-15ms after stimulation) or at 50-60ms when only NMDAR currents are observed. N=3. C) Whole-cell LTD recordings from two independent pathways in 10μM L689 in uninfected CA1 neurons; one pathway (filled symbols) received a 15-min 1-Hz low frequency stimulus (LFS), where indicated. The control pathway (open circles) received no stimulus. (Inset) Representative responses obtained before and after LFS. Error bars represent SEM, N=13. *p<0.05, paired t-test. D) As in C) but in neurons expressing NLS-mCherry + PSD-95. LFS produces no depression. N=13. E) PSD-95 overexpression blocks LFS-induced LTD in the presence of L689. Individual data points for each neuron are superimposed; N=13. *p< 0.05, unpaired t-test.

Figure 3:. PSD-95 overexpression does not block ion-flux dependent LTD

A) Whole-cell LTD recordings from two independent pathways in uninfected CA1 neurons; one pathway (filled symbols) received LFS, where indicated. The control pathway (open circles) received no stimulus. Error bars represent SEM, N=17, *p<0.05, paired t-test. (Inset) Representative responses obtained before and after LFS. Scale bars, 10ms, 10pA. B) As in A) but in neurons expressing NLS-mCherry + PSD-95. N=10; **p<0.01, paired t-test. C) PSD-95 overexpression does not significantly affect LFS-induced LTD. N>10, individual data points for each neuron are superimposed, non-significant (n.s.), unpaired t-test.

Figure 4:. PSD-95 expression levels in mice and rats

A) Representative immunofluorescence images of PSD-95 levels in the CA1 region of the hippocampus of mice and rats of indicated ages. Scale bar is 10μm. B) Quantification of PSD-95 immunofluorescence in animals of indicated ages. N = 2 slices/animal, 3 images/slice, 5 ROIs/image; **p<0.01, ***p<0.001; unpaired t-test. C) Representative immunoblots of PSD-95 and actin in mice and rat cerebrum. PSD-95 band was normalized by the corresponding actin band and this ratio was then normalized to P35 signal. N=4 (rats) N=6 (mice), individual data points for each experiment are superimposed; *p<0.05; unpaired t-test.