Synaptic plasticity regulated by phosphorylation of PSD-95 Serine 73 in dorsal CA1 is required for contextual fear extinction
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Summary: This timely study provides important and novel findings with regard to the role of PSD-95 protein in fear extinction formation and helps to advance our understanding of how dendritic changes in the hippocampus regulates fear maintenance. The findings should appeal to those interested in hippocampal function, fear and fear-related conditions, and extinction-based therapies. The major strengths of the paper lie in the use of a wide range of complementary technical approaches, and the significance of addressing specific molecular mediators of fear attenuation. Reasonable alternative explanations were identified for some of the key findings and the conclusions may not perfectly reflect the observations and experimental designs.
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Abstract
The ability to extinguish fearful memories is essential for survival. Accumulating data indicate that the dorsal CA1 area (dCA1) contributes to this process. However, the cellular and molecular basis of fear memory extinction remains poorly understood. Postsynaptic density protein 95 (PSD-95) regulates the structure and function of glutamatergic synapses. Here, using dCA1-targeted genetic and chemogenetic manipulations in vivo combined with PSD-95 immunostaining and 3D electron microscopy ex vivo , we demonstrate that phosphorylation of PSD-95 at serine 73 PSD-95(S73) is necessary for contextual fear extinction-induced expression of PSD-95 and synaptic plasticity. Moreover, PSD-95(S73) phosphorylation is not necessary for fear memory formation and recall but is required for extinction of contextual fear. Overall, our data shows how PSD-95-dependent synaptic plasticity in the hippocampus contributes to the persistence of fear memories.
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Reviewer #3:
In this manuscript, the authors investigated roles of PSD95 in the hippocampus for contextual fear extinction. The authors showed that PSD95 levels in the spine and density of PSD-95-positive spines in the dorsal CA1 (dCA1) are changed following contextual fear conditioning and extinction learning. Interestingly, overexpression of PSD95-S73A mutant or chemogenetic inhibition of dCA1 impairs only the second extinction learning at 24 hrs following the first extinction learning. Importantly, these manipulations also blocked the changes of PSD95-positive spines following the first extinction learning. These observations suggest that phosphorylation of PSD95 at S73 in the dCA1 of hippocampus contributes to contextual fear extinction. This manuscript suggests the importance of PSD95 phosphorylation in the hippocampus in some …
Reviewer #3:
In this manuscript, the authors investigated roles of PSD95 in the hippocampus for contextual fear extinction. The authors showed that PSD95 levels in the spine and density of PSD-95-positive spines in the dorsal CA1 (dCA1) are changed following contextual fear conditioning and extinction learning. Interestingly, overexpression of PSD95-S73A mutant or chemogenetic inhibition of dCA1 impairs only the second extinction learning at 24 hrs following the first extinction learning. Importantly, these manipulations also blocked the changes of PSD95-positive spines following the first extinction learning. These observations suggest that phosphorylation of PSD95 at S73 in the dCA1 of hippocampus contributes to contextual fear extinction. This manuscript suggests the importance of PSD95 phosphorylation in the hippocampus in some aspects of mechanisms of contextual fear extinction at the molecular and spine levels. However, the title, abstract and conclusions do not well reflect observations and experimental designs in this manuscript. I have several concerns as follows.
Major concerns:
The authors used viral overexpression of PSD-95 S73A mutant that may function as a dominant negative mutant, but not knock in mutation. Therefore, the function of phosphorylation of PSD 95 at S73 on spine morphology and contextual fear extinction have been not yet investigated well. The experimental design in this manuscript made limitations to understand behavioral results. It is better to use knock-in mutation strategy than overexpression of the mutant. Alternatively, the authors can examine the phosphorylation levels of PSD95 following contextual fear conditioning and extinction learning and/or function of this mutant at the molecular and cellular levels using biochemistry/molecular biology/cell culture.
Overexpression of S73A or chemogenetic inhibition of CA1 impaired additional extinction learning. These observations are interesting. However, the authors have not well characterized these findings at the behavioral levels. In other words, the authors should clarify the effects of these manipulations on contextual fear extinction at the behavioral levels. According to abundant knowledge of fear memory extinction, the behavioral results in this manuscript raised a lot of questions to understand the impact of those genetic manipulations on "contextual fear extinction". How about effects on extended extinction learning (60 min), additional 30 min extinction learning at the same day after first extinction training, spontaneous recovery, renewal, and reinstatement? Some answers of these questions will help to understand behavioral observations in this study and enable us to identify roles of PSD95 and its phosphorylation in extinction of contextual fear memory. It is also important to examine PSD95-positive spines just after the additional extinction learning to understand behavioral observations.
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Reviewer #2:
Ziółkowska et al. investigate synaptic processes in the dorsal hippocampal CA1(dCA1) region with the goal of testing the role of postsynaptic density protein 95 (PSD-95) dynamics in contextual fear extinction. They conclude that 1) extinction increases synaptic dCA1 PSD-95 levels and induces remodeling of dendritic spines, 2) extinction-related PSD-95 changes are mediated by phosphorylation of PSD-95 at serine 73, and 3) phosphorylation of PSD-95 at serine 73 as well as dCA1 activity are required to "update a partially extinguished fear memory". The experiments provide new insight and address a timely and important issue. The major strengths of the paper lie in the use of a wide range of complementary technical approaches, and the significance of addressing specific molecular mediators of fear attenuation. However, some of …
Reviewer #2:
Ziółkowska et al. investigate synaptic processes in the dorsal hippocampal CA1(dCA1) region with the goal of testing the role of postsynaptic density protein 95 (PSD-95) dynamics in contextual fear extinction. They conclude that 1) extinction increases synaptic dCA1 PSD-95 levels and induces remodeling of dendritic spines, 2) extinction-related PSD-95 changes are mediated by phosphorylation of PSD-95 at serine 73, and 3) phosphorylation of PSD-95 at serine 73 as well as dCA1 activity are required to "update a partially extinguished fear memory". The experiments provide new insight and address a timely and important issue. The major strengths of the paper lie in the use of a wide range of complementary technical approaches, and the significance of addressing specific molecular mediators of fear attenuation. However, some of the analysis is based on inadequately justified or inappropriate measures (e.g. that do not directly assay the phenomenon under investigation), and there are concerns about independent effects of viral overexpression in this system as well as the relevance of the behavioral analysis. The conclusions from the paper, if true, would appear to support a very intricate model involving PSD95 phosphorylation and synaptic accumulation after extinction, but because of weaknesses in the underlying evidence, these mechanisms and their relationship to extinction memory were not persuasively demonstrated. Following are some specific concerns:
The mean intensity of PSD95 labeling per spine appears to be affected in some hippocampal layers (Fig. 1), but this might be attributable in some cases to elimination of spines that have relatively lower PSD-95, rather than a change in PSD-95 levels, per se.
The quantification of overexpressed PSD-95 in Fig. 2 makes unclear what specifically has been measured. The methods suggest that % area is defined as the total area of mCherry labeling divided by the total image area. This is not a direct measure of PSD-95 levels, rather than morphological or protein localization changes. Furthermore, the localization of overexpressed PSD-95 (Fig. 2) is clearly very different from that of endogenous PSD-95 (Fig. 1) in that it accumulates throughout the dendrites. This makes it unclear what a "puncta" represents, or whether the analysis implies anything about synaptic function.
The authors argue that S73 phosphorylation is required for synapse elimination during extinction, but Fig. S2 (which is not referenced or discussed in the manuscript) and Fig. 3 indicate that the effect of S73A overexpression is to dramatically reduce spine density in both behavioral groups. It is therefore not clear whether the manipulation interacted with extinction to prevent spine removal, or simply occluded such an effect because spine density was already at an artificial floor prior to any behavioral training. Overexpression of the wildtype construct also reduced spine density to a similar degree. Furthermore, the S73A mutant protein dramatically increased PSD area (Fig. 3d), which apparently contradicts the notion that phosphorylation of this site is required for synaptic accumulation, when applying the same logic used elsewhere in the paper. These are serious confounding issues because the central claim of the paper is that S73 phosphorylation mediates PSD95 synaptic accumulation and synaptic strengthening.
The authors suggest that successive days of extinction represent a distinct process called updating of a partly extinguished memory, which they seem to imply has different molecular requirements. There appears to be no basis in the literature for this idea.
The analysis of extinction relies on measurement of within-session decreases in freezing. However, within-session extinction has been shown to be neither sufficient nor essential for between-session extinction. It is not even clear that within-session extinction is really even extinction at all, rather than, for example, habituation. It is essential to examine the retention of decreased freezing across days in order to establish that the formation of long-term memory is involved.
Finally, numerous comparisons are made between animals that received FC, with no further manipulation, and extinguished animals. This design leaves open the possibility that any differences are attributable not to an extinction process but instead to context exposure independent of fear regulation. A behavioral control in which animals receive context exposures, but no shocks, would be very useful.
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Reviewer #1:
Patients with posttraumatic stress disorder show impaired fear extinction that leads to persistent fear memories. The CA1 subregion of the hippocampus has been implicated in the acquisition and extinction of contextual fear memories, and both mechanisms depend on glutamatergic synaptic plasticity in this region. Postsynaptic density protein 95 (PSD-95) is known to regulate structural and functional changes in glutamatergic synapses, but whether PSD-95 participates in the acquisition and extinction of contextual fear memories remains unclear. To address this question, here Ziółkowska and coworkers used nanoscale-resolution analyses of PSD-95 protein in the CA1 combined with genetic and chemogenetic manipulations in mice exposed to a classical Pavlovian contextual fear conditioning paradigm. The study revealed that …
Reviewer #1:
Patients with posttraumatic stress disorder show impaired fear extinction that leads to persistent fear memories. The CA1 subregion of the hippocampus has been implicated in the acquisition and extinction of contextual fear memories, and both mechanisms depend on glutamatergic synaptic plasticity in this region. Postsynaptic density protein 95 (PSD-95) is known to regulate structural and functional changes in glutamatergic synapses, but whether PSD-95 participates in the acquisition and extinction of contextual fear memories remains unclear. To address this question, here Ziółkowska and coworkers used nanoscale-resolution analyses of PSD-95 protein in the CA1 combined with genetic and chemogenetic manipulations in mice exposed to a classical Pavlovian contextual fear conditioning paradigm. The study revealed that PSD-95-dependent synaptic plasticity in the dorsal CA1 area is not necessary for fear acquisition or the initial phase of fear extinction, but is critical for updating a partially extinguished fear memory. In addition, phosphorylation of PSD-95 at serine 73 is necessary for contextual fear extinction-induced PSD-95 expression and remodeling of dendritic spines in this region, suggesting a potential mechanism for fear memory persistence.
This timely study provides important and novel findings with regard to the role of PSD-95 protein in fear extinction formation and helps to advance our understanding of how dendritic changes in the hippocampus regulates fear maintenance. The present findings should be of general interest to the scientific community because extinction-based therapies are the gold-standard treatment for many fear-related disorders. The manuscript is clear, and the experiments were well-designed and executed. While the study is elegant, there are several important points including data interpretation that need to be clarified.
Major points:
- The authors identified changes in PSD-95 expression levels and spine density after both fear acquisition and fear extinction. Similarly, S73-dependent phosphorylation of PSD-95 and changes in spine density were also reported following both phases. How do the authors explain the lack of effects on fear acquisition and extinction after the infusion of S73-deficient PSD-95 expressing virus? Does this suggest that the observed dynamics of PSD-95 are not important for the fear memory expression? The interpretation of these findings should be clarified in the discussion.
Previous studies have demonstrated a key role of dorsal hippocampus CA1 area on fear retrieval and extinction acquisition using either lesion (e.g., Ji and Maren 2008, PMID: 18391185), or optogenetic tools (e.g., Sakagushi et al, 2015, PMID: 26075894). However, in the present study, chemogenetic inhibition of this same region had no effect on fear retrieval or extinction acquisition (Figures 5 and 6). How do the authors reconcile the lack of effects on fear retrieval and extinction acquisition with the previous literature? Similarly, previous studies on the role of hippocampal PSD-95 protein in extinction memory should be described and the main differences in the experimental design and findings should be discussed (e.g.; Nagura et al, 2012, PMID: 23268962; Cai et al, 2018; PMID: 30143658; Li et al 2017, PMID: 28888982)
The authors have used scanning electron microscopy to analyze the ultrastructure of dendritic spines and determine whether PSD-95 regulates extinction-induced synaptic growth. In addition, the authors complemented these studies by investigating the effect of PSD-95-overexpression and fear extinction training on synaptic transmission in the dorsal CA1 ex vivo. However, it is hard to understand what does the observed changes in dendritic spines and amplitude of EPSCs mean if the behavior of the animals was the same. This point should be discussed in the article.
In Figure 5, the authors showed that chemogenetic inactivation of CA1 changed PSD-95 expression in all the 3 subregions of CA1 (stOri, stRad and stLM). However, the extinction training behavior in Figure 1 demonstrated an effect only in 2 subregions (stOri and stLM). The authors should clarify this discrepancy. In addition, in the same series of experiments (Fig. 5Ciii), it is unclear whether the reduction in PSD-95 expression induced by chemogenetic inactivation is sufficient to bring the PSD-95 expression to the same post-conditioning levels.
The authors showed an interesting behavioral effect in the second part of the extinction phase (Figure 6C), similar to the results in Figure 4C. However, to confirm that phosphorylated PSD-95 is crucial for the maintenance of extinction memory, the authors may want to consider a direct comparison between the levels of phosphorylated PSD-95 right after extinction 1 and extinction 2. Differences in the expression would clarify whether the phosphorylated PSD-95 expression is further increased after additional extinction training, which would help to link the effect of chemogenetic inactivation on behavior. At least some discussion is needed for this part.
The authors used immunostaining and confocal tools to analyze 3 domains of dendritic tree of dorsal CA1 area in Thy1-GFP(M) mice (stOri, stRad and stLM) on different fear phases (conditioning and extinction). They found a significant decrease of PSD-95 expression, spine density and spine area in stOri and stRad during conditioning and a rescue of such decrease during extinction. However, the authors’ interpretation is that extinction resulted in an upregulation of PSD-95, which doesn't seem to be the case if you compare the numbers with the naïve group. Please clarify this point.
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Summary: This timely study provides important and novel findings with regard to the role of PSD-95 protein in fear extinction formation and helps to advance our understanding of how dendritic changes in the hippocampus regulates fear maintenance. The findings should appeal to those interested in hippocampal function, fear and fear-related conditions, and extinction-based therapies. The major strengths of the paper lie in the use of a wide range of complementary technical approaches, and the significance of addressing specific molecular mediators of fear attenuation. Reasonable alternative explanations were identified for some of the key findings and the conclusions may not perfectly reflect the observations and experimental designs.
Reviewer #1 opted to reveal their name to the authors in the decision letter after review.
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