Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy
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Curated by eLife
Evaluation Summary:
Synapses convey information in the brain, including signals from the environment. The changes in the incoming signals can alter the efficacy of synaptic transmission, which in turn can be represented by the changes in synaptic structure that is particularly evident in the postsynaptic compartment called spines. This study uses a custom-built superresolution microscope to follow individual spine shape and the dynamics of the resident scaffolding protein PSD95 simultaneously, to study the effects of rearing mice in an enriched environment relative to a simple standard cage. The imaging data are of superb quality. The authors find that regardless of the rearing condition, dynamic changes in the sizes of spine head and PSD95 are detected that do not necessarily correlate with each other. Furthermore, mice reared in an enriched environment show less variable spine head size. While these findings may be of potential interest to neuroscientists studying synaptic network architecture, a clarification of the biological question being addressed, and validation of the method used to monitor PSD95, would considerably strengthen the study and enhance its overall impact.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex. In general, the spine head geometry and PSD95 assemblies were highly dynamic, their changes depended linearly on their original size but correlated only mildly. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more uniformly. The topography of the PSD95 nanoorganization was more dynamic after environmental enrichment; changes in size were smaller but more correlated than in mice housed in standard cages. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.
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Author Response:
Reviewer #2 (Public Review):
The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.
Strengths
-The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.
-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.
-They…
Author Response:
Reviewer #2 (Public Review):
The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.
Strengths
-The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.
-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.
-They also find that there is less variability in the spine head size in animals in an enriched environment.
Weaknesses
-The authors use an enriched environment plasticity paradigm to showcase the method and measure spine head and PSD95 size and how they change over short periods of time. This particular biological study is not well-motivated and there is not a stated reason for studying the short-term (30-120 minutes) dynamics of PSD95 and spine head size, and their correlations. They also show that the variability in spine head size is decreased with the enriched environment, but do not show what the implications of that change would be from a biological point of view for synaptic dynamics or synaptic function.-The authors show that there are differences in the morphology of PSD95 between mice reared in enriched environments and those in control environments. While this quantification is done blindly by three different analysts, it is not done in a quantitative way. Also the authors do not show or explain the biological relevance of differences in the morphologies of PSD95, thus it is not clear what this measure means for synaptic plasticity or function.
-The authors use a cranial window preparation, which is commonly used in the literature. However, it is not clear how long they wait to image the mice after the cranial window. Previous work from Xu et al. (PMID: 17417634) suggests that there is in an increase in glial activation for a period of up to a month after surgery. The authors have not shown the degree of glial activation that follows after their surgeries and if they have not waited a month, there may be upregulation of microglia, which may alter synaptic stability (also demonstrated in the same paper). The authors have not discussed this point or the implications for their findings.
We thank the reviewer for his/her valuable input.
The time-scale we study is similar to what is known from structural changes after LTP and thus we wanted to study the same time scale in vivo. We revised the motivation and explained better the biological relevance of the observed changes. We absolutely agree with the reviewer on his/her concern for chronic imaging. However, we performed acute experiments and imaged directly after implanting the window in the same session. After imaging the mice were sacrificed.
Reviewer #3 (Public Review):
Wegner et al. use two-color STED to follow spines and their PSDs in layer1 of mouse visual cortex over 2 hours under anesthesia. They compare mice that were kept in an enriched environment (EE) to control mice housed in standard laboratory cages. Spines in EE mice are larger and show larger fluctuations in size. PSDs in EE mice shrink during anesthesia and tend to change their nanostructure. Very importantly, changes in spine size were not driven by PSD size changes, or vice versa. Technologically, this is a landmark study, as tracking two different labeled structures in individual synapses at the nanoscale can obviously be applied to a large number of synaptic proteins and organelles, two at a time. Single-color superresolution microscopy is much less useful, as 'puncta in space', without cellular context, are difficult to interpret. This pioneering work is the first proof-of-concept of two-color in-vivo STED and of major importance for the community. Although stochastic processes seem to drive much of the synaptic dynamics under anesthesia, the environment shapes the spine size distribution and affects synaptic dynamics in a lasting fashion.
One major comment:
l.259: "These results suggest that Ctr housed mice undergo stronger morphological changes." This I find a bit misleading. What about: These results suggest that anesthesia induces stronger morphological changes in Ctr housed mice? Altogether, a discussion of the potential effects of anesthesia on spine/PSD dynamics is missing (see e.g. Yang et al., DOI: 10.1371/journal.pbio.3001146). The fact that there was weak correlation between spine head and PSD fluctuation could have something to do with the state of suppressed activity the system was in during imaging. Under conditions of intense processing of visual information, changes might have been more rapid and more tightly correlated. This could be mentioned as a perspective for the future - to visually stimulate the anesthetized animal.
We agree with the reviewer that it should be mentioned here that the morphological change was observed under anesthesia. However, the sentence suggested by the reviewer is also a bit misleading since it suggests that the anesthesia has triggered the change. We think that anesthesia might affect the amplitude and dynamic of the observed changes but does not induce the change. Thus we rephrased as follows: These results suggest that Ctr housed mice undergo stronger morphological changes under anesthesia.
We absolutely agree about the potential influence of the anesthesia on the spine and PSD95 nanoplasticity and added the following comment. Of course, we would like to perform the measurement in the future also in awake mice and after visual stimulation.
Added to discussion: However, it was shown that MMF anesthesia reduces spiking activity and mildly increases spine turnover in the hippocampus (Yang et al., 2021). Thus, the plasticity of spine heads and PSD95 assemblies might be different in the awake state and under intense processing of visual information.
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Evaluation Summary:
Synapses convey information in the brain, including signals from the environment. The changes in the incoming signals can alter the efficacy of synaptic transmission, which in turn can be represented by the changes in synaptic structure that is particularly evident in the postsynaptic compartment called spines. This study uses a custom-built superresolution microscope to follow individual spine shape and the dynamics of the resident scaffolding protein PSD95 simultaneously, to study the effects of rearing mice in an enriched environment relative to a simple standard cage. The imaging data are of superb quality. The authors find that regardless of the rearing condition, dynamic changes in the sizes of spine head and PSD95 are detected that do not necessarily correlate with each other. Furthermore, mice reared in an …
Evaluation Summary:
Synapses convey information in the brain, including signals from the environment. The changes in the incoming signals can alter the efficacy of synaptic transmission, which in turn can be represented by the changes in synaptic structure that is particularly evident in the postsynaptic compartment called spines. This study uses a custom-built superresolution microscope to follow individual spine shape and the dynamics of the resident scaffolding protein PSD95 simultaneously, to study the effects of rearing mice in an enriched environment relative to a simple standard cage. The imaging data are of superb quality. The authors find that regardless of the rearing condition, dynamic changes in the sizes of spine head and PSD95 are detected that do not necessarily correlate with each other. Furthermore, mice reared in an enriched environment show less variable spine head size. While these findings may be of potential interest to neuroscientists studying synaptic network architecture, a clarification of the biological question being addressed, and validation of the method used to monitor PSD95, would considerably strengthen the study and enhance its overall impact.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study capitalizes on the crosstalk-free two-color STED developed by the authors (cf. Willg et al., Cell Rep 2021) to examine the dynamic changes in synapse structure in mouse visual cortex. Imaging the superficial dendrites of layer V pyramidal neurons, the authors report features of dendritic spine morphology and the dynamics of scaffolding protein contained within that are affected by rearing mice in an enriched environment (EE) compared to control housing. Curiously, EE mice show less variable spine head volumes and PSD95 areas compared to control mice, while the spine head volume is larger but not PSD95 area in EE group compared to the control group. Moreover, nano-organization of PSD95 displays more prominent changes in EE compared to controls. These findings may be of potential interest to …
Reviewer #1 (Public Review):
This study capitalizes on the crosstalk-free two-color STED developed by the authors (cf. Willg et al., Cell Rep 2021) to examine the dynamic changes in synapse structure in mouse visual cortex. Imaging the superficial dendrites of layer V pyramidal neurons, the authors report features of dendritic spine morphology and the dynamics of scaffolding protein contained within that are affected by rearing mice in an enriched environment (EE) compared to control housing. Curiously, EE mice show less variable spine head volumes and PSD95 areas compared to control mice, while the spine head volume is larger but not PSD95 area in EE group compared to the control group. Moreover, nano-organization of PSD95 displays more prominent changes in EE compared to controls. These findings may be of potential interest to neuroscientists studying synaptic network architecture.
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Reviewer #2 (Public Review):
The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.
Strengths
-The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.
-They also find that there is …
Reviewer #2 (Public Review):
The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.
Strengths
-The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.
-They also find that there is less variability in the spine head size in animals in an enriched environment.
Weaknesses
-The authors use an enriched environment plasticity paradigm to showcase the method and measure spine head and PSD95 size and how they change over short periods of time. This particular biological study is not well-motivated and there is not a stated reason for studying the short-term (30-120 minutes) dynamics of PSD95 and spine head size, and their correlations. They also show that the variability in spine head size is decreased with the enriched environment, but do not show what the implications of that change would be from a biological point of view for synaptic dynamics or synaptic function.-The authors show that there are differences in the morphology of PSD95 between mice reared in enriched environments and those in control environments. While this quantification is done blindly by three different analysts, it is not done in a quantitative way. Also the authors do not show or explain the biological relevance of differences in the morphologies of PSD95, thus it is not clear what this measure means for synaptic plasticity or function.
-The authors use a cranial window preparation, which is commonly used in the literature. However, it is not clear how long they wait to image the mice after the cranial window. Previous work from Xu et al. (PMID: 17417634) suggests that there is in an increase in glial activation for a period of up to a month after surgery. The authors have not shown the degree of glial activation that follows after their surgeries and if they have not waited a month, there may be upregulation of microglia, which may alter synaptic stability (also demonstrated in the same paper). The authors have not discussed this point or the implications for their findings.
-
Reviewer #3 (Public Review):
Wegner et al. use two-color STED to follow spines and their PSDs in layer1 of mouse visual cortex over 2 hours under anesthesia. They compare mice that were kept in an enriched environment (EE) to control mice housed in standard laboratory cages. Spines in EE mice are larger and show larger fluctuations in size. PSDs in EE mice shrink during anesthesia and tend to change their nanostructure. Very importantly, changes in spine size were not driven by PSD size changes, or vice versa. Technologically, this is a landmark study, as tracking two different labeled structures in individual synapses at the nanoscale can obviously be applied to a large number of synaptic proteins and organelles, two at a time. Single-color superresolution microscopy is much less useful, as 'puncta in space', without cellular context, …
Reviewer #3 (Public Review):
Wegner et al. use two-color STED to follow spines and their PSDs in layer1 of mouse visual cortex over 2 hours under anesthesia. They compare mice that were kept in an enriched environment (EE) to control mice housed in standard laboratory cages. Spines in EE mice are larger and show larger fluctuations in size. PSDs in EE mice shrink during anesthesia and tend to change their nanostructure. Very importantly, changes in spine size were not driven by PSD size changes, or vice versa. Technologically, this is a landmark study, as tracking two different labeled structures in individual synapses at the nanoscale can obviously be applied to a large number of synaptic proteins and organelles, two at a time. Single-color superresolution microscopy is much less useful, as 'puncta in space', without cellular context, are difficult to interpret. This pioneering work is the first proof-of-concept of two-color in-vivo STED and of major importance for the community. Although stochastic processes seem to drive much of the synaptic dynamics under anesthesia, the environment shapes the spine size distribution and affects synaptic dynamics in a lasting fashion.
One major comment:
l.259: "These results suggest that Ctr housed mice undergo stronger morphological changes." This I find a bit misleading. What about: These results suggest that anesthesia induces stronger morphological changes in Ctr housed mice? Altogether, a discussion of the potential effects of anesthesia on spine/PSD dynamics is missing (see e.g. Yang et al., DOI: 10.1371/journal.pbio.3001146). The fact that there was weak correlation between spine head and PSD fluctuation could have something to do with the state of suppressed activity the system was in during imaging. Under conditions of intense processing of visual information, changes might have been more rapid and more tightly correlated. This could be mentioned as a perspective for the future - to visually stimulate the anesthetized animal. -
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