Durable fear memories require PSD-95

Abstract

Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95(GK)), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95(GK) mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

Figure 1. PSD-95 deletion does not affect retrieval of recent fear memories, but impairs their extinction and precision

(a) Schematic of experimental design for testing recent fear memory retrieval following delay conditioning. (b) Genotypes showed equivalent levels of freezing during cue and context retrieval (n=11–17). (c) Schematic of experimental design for testing recent fear memory retrieval following trace conditioning. (d) Following trace fear conditioning, genotypes showed similar cue fear retrieval, but PSD-95^GK mice froze significantly less than wild-type controls during context retrieval (n=8–10). (e) Schematic of experimental design for testing extinction of a recent fear memory. (f) WT controls, but not PSD-95^GK mice, decreased freezing from the first to the last extinction trial-block, and froze less during extinction retrieval as compared to the first trial-block of extinction training (n=11). (g) Schematic of experimental design for testing the precision of a recent contextual fear memory. (h) WT controls, but not PSD-95^GK mice, froze more to the conditioned context than either of two different contexts (n=13). Data are means ± SEM. *P<.05 vs. WT/Context, #P<.05 vs. 1st Ext-block/WT, †P<.05 vs. Ren/WT, ‡P<.05 vs. Context A/WT

Figure 2. PSD-95 deletion impairs retrieval and stability of remote fear memories

(a) Schematic of experimental design for testing remote retrieval of a cued fear memory. (b) PSD-95^GK mice froze less than WT controls during remote fear memory retrieval (n=8–9). (c) Schematic of experimental design for testing the remote retrieval of a contextual fear memory. (d) PSD-95^GK mice froze less than WT controls during remote fear memory retrieval (n=9–10). (e) Schematic of experimental design for testing recent and remote retrieval of a contextual fear memory in the same mice. (f) PSD-95^GK mice froze less than WT controls during remote, but not recent, fear memory retrieval (n=8). Data are means ±SEM. *P<.05 vs. WT/same time point, †P<.05 vs. non-reactivated MUT

Figure 3. PSD-95 deletion disrupts activation of the infralimbic cortex during remote fear memory retrieval

(a) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. (b) PSD-95^GK mice froze less than WT controls during remote, but not recent, fear memory retrieval. (c) Representative Zif268-labelled coronal section showing the three prefrontal regions analyzed. (d) IEG analysis of prefrontal regions found no change in the number of Zif268-positive cells in either the anterior cingulate cortex (ACC, Cg1 subregion) or prelimbic cortex (PL), irrespective of memory age or genotype. (e) WT mice exhibited an elevated number of Zif268-positive cells in the infralimbic cortex (IL) after remote, as compared to recent retrieval, while the number of cells did not increase in the PSD-95^GK mice. (f) Representative coronal sections showing Zif268-positive cells in the IL (scale bar= 150 µm). (g) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KO (top) and WT controls (bottom) (scale bar=15 µm). (h) PSD-95 KO mice had marginally lesser density and significantly lesser spine head width on IL pyramidal neurons, relative to WT controls (n=3 mice per group, n=7–12 cells per group). Data are means ±SEM. *P<.05 vs. WT Remote or WT controls, #P<.05 vs. WT Recent

Figure 4. PSD-95 deletion disrupts CS-related single-unit activity in the infralimbic cortex during fear memory retrieval

(a) PSD-95^GK mice showed less CS-related IL single-unit activity than WT controls during recent (left panel) and remote (right panel) fear memory retrieval. Insets show first 100 millisecond timebins. (b) PSD-95^GK mice had fewer phasic CS-related IL single-units than WT controls during recent and remote retrieval. (c) IL single-unit activity correlated positively with freezing during remote retrieval. (d) IL single-unit burst activity increased during the CS in both genotypes during recent retrieval, but only in WT mice during remote retrieval. (e) PSD-95^GK mice showed lower gamma and theta oscillations than WT controls, on recent and remote retrievals. (f) Representative perievent spectrograms showing the gamma and theta frequency spectra around CS-onset. For gamma panels, darkest blue=0 mV²*s and darkest red=2e⁻⁵. For theta panels, darkest blue=0 and darkest red=4e⁻⁴ mV²*s. Data are means ±SEM. n=6 mice per genotype, n=74–87 single-units per genotype. *P<.05 vs. WT/same time point, #P<.05 vs. pre-CS/same genotype

Figure 5. PSD-95 knockdown in infralimbic cortex impairs remote fear memory retrieval

(a) Example of green fluorescent protein (GFP) localization of the PSD-95 knockdown adenoassociated virus in the IL (scale bar=200 µm). (b) Example Western blots showing viral-induced knockdown of PSD-95 protein. (c) Schematic of experimental design for testing recent and remote retrieval of a cued fear memory. (d) IL PSD-95 KD mice showed less freezing than GFP-virus controls during remote, but not recent retrieval (n=12–17). (e) ACC PSD-95 KD mice showed similar freezing levels to GFP controls during recent and remote retrieval (n=9–11). Inset: example of GFP localization of the PSD-95 KD adenoassociated virus in the Cg1 region of the ACC (scale bar=200 µm). (f) Schematic of experimental design for testing fear extinction. (g) IL PSD-95 KD mice decreased freezing from the first to the last extinction trial-block, but less so than GFP-virus controls. GFP-virus controls, but not IL PSD-95 KD mice, froze less during extinction retrieval as compared to the first trial-block of extinction training (n=7–9). (h) Example images of dendritic spines in IL pyramidal neurons from a PSD-95 KD (top) and GFP control (bottom) (scale bar=15 µm). (i) Examples and cartoon showing examples of narrow (N) and wide (W) subpopulations of dendritic spines. (j,k) PSD-95 KD decreased the spine head width, but not density, of relatively wide spines on IL pyramidal neurons, relative to GFP controls (n=7 mice per group, n=15–23 cells per group). Data are means ±SEM. *P<.05 vs. PSD-95 KD/same time point or spine subpopulation