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. 2007 Mar 6;104(10):4176-81.
doi: 10.1073/pnas.0609307104. Epub 2007 Feb 27.

PSD-95 is required for activity-driven synapse stabilization

Ingrid Ehrlich  1 , Matthew Klein, Simon Rumpel, Roberto Malinow
Affiliations

PSD-95 is required for activity-driven synapse stabilization

Ingrid Ehrlich et al. Proc Natl Acad Sci U S A. .

Abstract

The activity-dependent regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and the stabilization of synapses are critical to synaptic development and plasticity. One candidate molecule implicated in maturation, synaptic strengthening, and plasticity is PSD-95. Here we find that acute knockdown of PSD-95 in brain slice cultures by RNAi arrests the normal development of synaptic structure and function that is driven by spontaneous activity. Surprisingly, PSD-95 is not necessary for the induction and early expression of long-term potentiation (LTP). However, knockdown of PSD-95 leads to smaller increases in spine size after chemically induced LTP. Furthermore, although at this age spine turnover is normally low and LTP produces a transient increase, in cells with reduced PSD-95 spine turnover is high and remains increased after LTP. Taken together, our data support a model in which appropriate levels of PSD-95 are required for activity-dependent synapse stabilization after initial phases of synaptic potentiation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Knockdown of PSD-95 decreases synaptic strength. (a–c) EPSCs recorded from pairs of CA1 pyramidal neurons at −60 or +40 mV (one control and one expressing shRNA). (a and b) Hp1 and Hp2 decreased AMPA-EPSCs and, to a lesser extent, NMDA-EPSCs. (c) Scr1 had no effect on transmission. (Scale bars: 50 pA and 40 ms.) (d) For Hp1 and Hp2, AMPA-EPSCs decreased to 57.9 ± 11.4% (n = 22, P < 0.001) and to 70.2 ± 9.4% of control (n = 12, P < 0.05); there was no change for Scr1 (107.6 ± 14.0%, n = 14, P = 0.83). (e) For Hp1 and Hp2, NMDA-EPSCs decreased to 71.1 ± 13.3% (n = 24, P = 0.02) and to 75.6 ± 15.1% of control (n = 11, P = 0.01); there was no change for Scr1 (92.3 ± 20.2%, n = 14, P = 0.68).
Fig. 2.
Fig. 2.
Knockdown of PSD-95 prevents the developmental increase in synapses with functional AMPA-Rs. (a) AMPA-mEPSCs recorded at −60 mV from CA1 neurons at 6 Div and 9 Div (one control and one expressing Hp1 for 3 days). (Scale bars: 20 pA and 50 ms.) (b) The increase in mEPSC frequency from 0.36 ± 0.06 Hz (n = 13) to 0.83 ± 0.12 Hz (n = 11) (P < 0.001) was prevented by Hp1 (0.46 ± 0.06 Hz, n = 11, P = 0.01 vs. 9 Div). (c) There was no difference in average mEPSC amplitude in a cell-wise comparison (6 Div: 10.7 ± 0.2 pA, n = 13; 9 Div Con: 11.0 ± 0.5 pA, n = 11; 9 Div Hp1: 9.9 ± 0.6 pA, n = 11; P > 0.15).
Fig. 3.
Fig. 3.
Knockdown of PSD-95 arrests spine morphological development. (a) Two-photon images of secondary apical dendrites at 7 Div, and at 10 Div after expressing control or effective shRNA for 3 days. (Scale bar: 2 μm.) (b) Hp1 prevented the increase in spine density (∗, P < 0.01). Primary dendrites: 0.55 ± 0.03 (7 Div), 0.84 ± 0.04, and 0.54 ± 0.05 spines per micrometer (Con and Hp1 at 10 Div). Secondary dendrites: 0.55 ± 0.02 (7 Div), 0.84 ± 0.04, and 0.62 ± 0.05 spines per micrometer (Con and Hp1 at 10 Div). Tertiary dendrites: 0.53 ± 0.02 (7 Div), 0.72 ± 0.05, and 0.52 ± 0.04 spines per micrometer (Con and Hp1 at 10 Div). No difference was observed between 7 Div and Hp1 at 10 Div (P > 0.13). (c) Hp1 prevented changes in spine type (∗, P < 0.05). Fraction of stubby spines: 0.43 ± 0.01 (7 Div), 0.30 ± 0.03, and 0.44 ± 0.02 (Con and Hp1 at 10 Div). Fraction of mushroom spines: 0.33 ± 0.02 (7 Div), 0.40 ± 0.02, and 0.32 ± 0.03 (Con and Hp1 at 10 Div). The fraction of thin spines did not change (P > 0.16). (d) Hp1 prevented spine size changes (∗, P < 0.04). Average spine sizes were 59.8 ± 7.9 (7 Div), 84.1 ± 9.7 (Con 10 Div), and 61.1 ± 4.7 (Hp1 10 Div; P = 0.44 vs. 7 Div) in arbitrary units (AU). (Right) Cumulative distributions of spine sizes. n = 6 cells per group.
Fig. 4.
Fig. 4.
Decrease in synaptic strength by knockdown of PSD-95 is occluded by activity blockade. (a–c) EPSCs recorded from pairs of CA1 neurons at −60 or +40 mV after incubation in high Mg2+, TTX, or APV for 2–3 days showed no change, except for NMDA-EPSCs after incubation in APV. (Scale bars: 20 pA and 40 ms.) (d) In Hp1-expressing neurons, AMPA-EPSCs were 99.8 ± 15.3% (34.1 ± 6.0 vs. 34.0 ± 5.2 pA, n = 13, P = 0.98) for TTX-treated neurons, 85.0 ± 12.7% (34.7 ± 3.1 vs. 29.5 ± 3.8 pA, n = 17, P = 0.23) for Mg2+-treated neurons, and 100.7 ± 10.9% of control (27.2 ± 2.2 vs. 27.4 ± 3.0 pA, n = 12, P = 0.96) for APV-treated neurons. (e) In Hp1-expressing neurons, NMDA-EPSCs were 91.6 ± 21.1% (18.3 ± 2.5 vs. 16.7 ± 3.5 pA, n = 12, P = 0.38) for TTX-treated neurons, 101.5 ± 16.7% (17.7 ± 2.8 vs. 18.0 ± 3.0 pA, n = 12, P = 0.93) for Mg2+-treated neurons, and 68.2 ± 13.6% of control (15.1 ± 1.9 vs. 10.2 ± 1.5 pA, n = 10, P = 0.02) for APV-treated neurons.
Fig. 5.
Fig. 5.
Knockdown of PSD-95 affects morphological but not early functional changes during LTP. (a–e) Normal levels of PSD-95 are not required for induction and early expression of LTP. (a) No difference in LTP between control (nontransfected, n = 10) and Hp1-expressing (n = 8) (P = 0.23) neurons. (c) LTP in control and Hp1-expressing neurons was sensitive to the CaMKII blocker KN-93 applied 20 min before and during induction. (b and d) Average EPSCs before and 30–35 min after pairing. (Scale bars: 20 pA and 20 ms.) (e) Summary of all LTP data: When preincubation in TTX was used to prevent decrease in NMDA-EPSCs, no difference in LTP was observed (nontransfected: 2.46 ± 0.31, n = 7; Scr1: 2.10 ± 0.37, n = 7; Hp1: 2.27 ± 0.48, n = 8; P > 0.46). Under normal conditions, LTP was 1.9 ± 0.14 (n = 10) in control neurons and blocked by KN-93 (1.31 ± 0.21, n = 9, P < 0.03). In Hp1 neurons, LTP was 2.26 ± 0.28 (n = 8) and blocked by KN-93 (1.23 ± 0.21, n = 7, P < 0.02). (f and g) Knockdown of PSD-95 affects spine size changes after cLTP. (f) Spine changes (arrowheads) after cLTP in a control and a Hp1-neuron. (Scale bars: 1 μm.) (g) cLTP led to smaller increases in spine volume in Hp1 (26 ± 4%, n = 3 cells, 216 spines) than in control neurons (46 ± 7%, n = 3 cells, 171 spines) (P < 0.04).
Fig. 6.
Fig. 6.
Knockdown of PSD-95 increases spine turnover and transient spines. (a) Dendrite of Hp1 neuron imaged 30–90 min after cLTP showing extension and retraction of protrusions (arrows). (Scale bar: 2 μm.) (b) Spine TOR before and after cLTP increased in Hp1 vs. control neurons (∗, P < 0.04; n = 4 cells). In control neurons, TOR increased transiently after cLTP (∗∗, P < 0.05). (c) Fraction of transient spines (lifetime < 1 h) after cLTP was increased in Hp1 vs. control neurons (0.010 ± 0.002 vs. 0.054 ± 0.006 spines per micrometer per 30 min, n = 4 cells, P < 0.01). Transient spines were smaller (normalized to mean stable spine size; Con: 0.65 ± 0.10, n = 25; Hp1: 0.57 ± 0.04, n = 69). (d) Dendrite of a control neuron (12 Div) showing transient protrusions (arrows). (Scale bar: 2 μm.) (e) Basal TOR decreased over development and remained high after knockdown (12 Div: 0.063 ± 0.006, n = 3; 17 Div Con: 0.013 ± 0.007, n = 5; 17 Div Hp1: 0.076 ± 0.016 spines per micrometer per 30 min, n = 4 cells; ∗, P < 0.006). (f) The fraction of transient spines decreased but was elevated in Hp1 vs. control neurons (12 Div: 0.043 ± 0.006, n = 3; 17 Div Con: 0.008 ± 0.006, n = 5; 17 Div Hp1: 0.071 ± 0.004 spines per micrometer per 30 min, n = 4 cells; ∗, P < 0.012). Transient spines were smaller (12 Div: 0.55 ± 0.06, n = 29; Hp1: 0.44 ± 0.05, n = 25).
Fig. 7.
Fig. 7.
Knockdown of PSD-95 impairs LTD. (a) Synaptic depression after pairing-induced LTD was not detected in Hp1-expressing neurons. (b) Average AMPA-EPSCs at −60 mV recorded before and 30–35 min after LTD induction. (Scale bars: 20 pA and 20 ms.) (c) Summary of changes after LTD: In control neurons, EPSCs decreased to 0.51 ± 0.08 of baseline (n = 11), significantly different from Hp1 (0.98 ± 0.18, n = 7, P < 0.02). Control pathways were stable (0.92 ± 0.16 and 0.98 ± 0.19).
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