1. Author Response:

    Reviewer #1:

    This manuscript describes a novel tool for tracking synaptic plasticity at the single synapse resolution with a SEP-tagged GluA1 receptor. The authors rather convincingly demonstrate that this tool does not disturb synaptic physiology or mouse behavior. They also show that this tool can be used to measure the distribution of synaptic weights and its variation during a plasticity protocol in barrel cortex. This tool is useful for more quantitative measurements of synaptic strength in vivo. The main weakness of the method is related to the density of marked synapses which makes the tracking difficult with only 80% reproducibility, probably due to the resolution limits of 2P-microscopy. It could however be improved with in vivo superresolution technique. The other limit of the method is that it is not demonstrated to allow longitudinal studies at the single synapse resolution. The authors do not discuss this issue in detail. It seems feasible with additional markers of the dendrite and spines. But this is not developed in the manuscript. Also, it is not demonstrated to which extent this technique outperforms traditional methods for synaptic weight measurements like spine volume.

    We have included new experiments and analyses to further demonstrate the utility of these tools and expanded discussion of the limits of our approach to automatic synapse detection. We are further demonstrating that by introducing a red cytosolic fluorescent protein, longitudinal imaging of the same synapses across days is achievable with this mouse line. We further discuss how imaging SEP-GluA1 is a more accurate readout of synaptic plasticity and strength than spine volume, since synaptic strength is mediated by receptors rather than spine size. In previous studies, we have shown that imaging sparsely expressed SEP-GluA1 can reveal plasticity at synapses that were not detectable by measuring spine size (Zhang et al., 2015) or reveal larger amplitudes of changes in SEP-GluA1 than spine size (Tan et al. 2020, Roth et al., 2020). The dissociation of spine size and synaptic strength has been reported many times (Lee et al., 2012). For instance, spine number or volume is not changed at all at cerebellar Purkinje cell synapses during LTD (Sdrulla and Linden, 2007) and Insulin-induced endocytosis of AMPARs is not accompanied by spine shrinkage (Wang et al., 2007). Thus, spine size, in certain conditions, is not a good indication of synaptic strength, Together, these experiments demonstrate that measuring SEP-GluA1 is a reliable and sensitive readout of synaptic strength and plasticity. We believe that our added data and discussion as suggested by the reviewers has improved and strengthened our demonstration of this SEP-GluA1 KI mouse line.

    Reviewer #2:

    Over the last couple of decades, the development of fluorescent transgenic mouse lines (e.g thy1-GFP) and delivery techniques (e.g., in utero electroporation), as well as the democratization of recording methods in living animals (such as calcium imaging, high-density probes,) have strengthened the link between synaptic plasticity and behavior. Nevertheless, these methods are most of the time limited to hundreds of cells at best (very few in the case of patch-clamp recordings), or failed to achieve a clear synaptic resolution without affecting the tight equilibrium of endogenous proteins.

    In this paper, Graves, Roth, Tan, Zhu, Bygrave, Lopez-Ortega et al present an additional high-resolution optical tool to overcome these limitations. They generated a new knock-in mouse line that fluorescently labels all endogenous AMPA receptors (KI SEP-GluA1). In this mouse line, the extracellular N-terminal domain of the GluA1 subunit of AMPAR is tagged with super ecliptic pHluorin (SEP), a pH sensitive variant of GFP that fluoresces at neutral pH (at cell surface) and is quenched at acidic pH (within the cell). This tool thus avoids the use of antibodies or the over-expression of exogenous tagged receptors.

    They perform a set of convincing experiments showing that synaptic transmission, homeostatic and activity-dependent synaptic plasticity in vitro (Figs2-3), and behavior (Fig. 4), are not affected in KI SEP-GluA1 as compared to wild-type mice. Despite the obvious quality and viability of this mouse line (this is an important tool with no doubt), it is puzzling however that, while the level of GluA2 remains unchanged, the global expression of SEP-GluA1 is twice as low as the expression of GluA1 in wild-type mice (Fig.1). The manuscript would benefit from a clear brain-region specific comparison between the expression pattern of GluA1 and SEP-GluA1. At least, the author should discuss this point, and how this might affect the formation of GluA1/A2 heteromers, the dominant form of AMPAR in pyramidal neurons.

    Then, they provide strong evidence that SEP-GluA1 receptors are mobile in vivo (Fig. 6) and can thus accurately report synaptic plasticity, at least in anesthetized animals (Figs. 7-8). The authors make a point that "this novel SEP-GluA1 knockin mouse is the first tool that enables longitudinal tracking of synaptic plasticity underlying behavior at brain-wide scale with single-synapse resolution". However, given the high density of fluorescent synapses, it remains unclear how effective would be this mouse line in awake mice, and more specifically during behavior, in which movement artifacts could preclude the tracking and registration of the same population of SEP-GluA1 containing synapses over time.

    Finally, they present a new automated analytical tool to detect and register fluorescent synapses (Fig.7). Although the initiative is important and laudable (it is true that imaging approaches are usually plagued by the lack of user-friendly analysis tools), the method would benefit from a comparison with existing methods.

    We thank the reviewer for their detailed evaluation of our manuscript and appreciate their insightful comments. Addressing the suggestions raised by the reviewer, we have now included new data and expanded our discussion which we believe has significantly improved our manuscript. As suggested, we now show that SEP-GluA1 expression levels are consistent across brain regions, discuss the ability to detect and track individual synapses in awake mice, and expanded the description of our detection algorithm to include comparisons with existing methods.

    Reviewer #3:

    Understanding the distribution of synaptic strength and plasticity in the brain is paramount for understanding neural circuit function underlying behavior. In the manuscript by Graves et al., the authors developed a novel mouse model for optical detection of synaptic strength and plasticity in live brains. Specifically, they modified the mouse genome by modifying the native GluA1 AMPAR subunit gene (gria1) with a pH-sensitive GFP (pHluorin) -tagged GluA1 at its N-terminus. This sensor is nearly maximally fluorescent when the pH is neutral and quenched in acidic environments and, therefore, preferentially marks AMPARs located on the plasma membrane. Since AMPARs are known to cluster in the postsynaptic density, AMPAR number is the predominant postsynaptic determinant of synaptic strength. Specifically, the trafficking of GluA1 AMPARs is responsible for LTP in CA1 hippocampus. The use of this novel genetic tool raises the possibility of monitoring synaptic strength optically, thus providing a strategy for massively parallel assessment of the distribution of synaptic strength in bulk brain tissue. Even more promising is the use of cranial windows and 2-photon microscopy to assess synaptic strength longitudinally, for example, during learning acquisition.

    The authors performed a comprehensive and rigorous set of control experiments using electrophysiology and behavior experiments. They demonstrated that modified GluA1 acts as native receptors and does not suffer from the shortcomings of overexpression approaches. The authors convincingly demonstrate that the modified receptors generate normal wild-type synaptic physiology and no behavioral alterations. Using glutamate uncaging, they showed that fluorescence changes at synapses were highly correlated with an electrophysiological assessment of synaptic strength and plasticity. Thus the data support claims that synaptic strength and plasticity could be assessed and monitored at unprecedented parallelization.

    Whether SEP-GluA1 can be used to quantify synaptic strength and its changes is uncertain due to an unknown ratio of GluA1/2 versus GluA2/3 receptors, the differential expression of GluA1 in different cell types, and the presence of GluA1-independent plasticities. Another potential shortcoming of the study is the lack of a ground truth demonstration of true synapses in vivo. Given the high-density of synapses, low z-resolution 2P microscopy (> 2 um), and the presence of a significant extrasynaptic pool, a confirmation of their results with superresolution or EM would be essential. Moreover, the lack of cell-type-specific labeling is likely to limit the tool's use for linking behavioral and microcircuit synaptic plasticity. It is possible that control experiments in acute brain slices could circumvent some shortcomings and provide a more quantitative workflow.

    We would like to thank the reviewer for their careful reading and evaluation of our manuscript and for providing valuable comments and recommendations. We have now added new data and expanded our discussion in response to the reviewer’s recommendation and believe that his has improved our manuscript. Among other points, we have added discussion regarding our mouse line’s ability to detect changes in GluA1 containing AMPARs, included measurements of the PSF of our microscope to quantify the detection limit of individual synapses with our approach, and demonstrated how the SEP-GluA1 knockin line can be used for measuring cell-type-specific changes in GluA1.

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  2. Evaluation Summary:

    This manuscript describes a novel tool for tracking synaptic plasticity at the single synapse resolution with a SEP-tagged GluA1 receptor expressed in a transgenic mouse. The authors rather convincingly demonstrate that this tool does not disturb synaptic physiology or mouse behavior. They also show that this tool can be used to measure the distribution of synaptic weights and its variation during a plasticity protocol in barrel cortex. This tool is useful for more quantitative measurements of synaptic strength in vivo, although some revisions would help making a convincing case of the usefulness of this tool with respect to previous methods. Genetic specificity of the expression of the construct is also a concern.

    (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. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This manuscript describes a novel tool for tracking synaptic plasticity at the single synapse resolution with a SEP-tagged GluA1 receptor. The authors rather convincingly demonstrate that this tool does not disturb synaptic physiology or mouse behavior. They also show that this tool can be used to measure the distribution of synaptic weights and its variation during a plasticity protocol in barrel cortex. This tool is useful for more quantitative measurements of synaptic strength in vivo. The main weakness of the method is related to the density of marked synapses which makes the tracking difficult with only 80% reproducibility, probably due to the resolution limits of 2P-microscopy. It could however be improved with in vivo superresolution technique. The other limit of the method is that it is not demonstrated to allow longitudinal studies at the single synapse resolution. The authors do not discuss this issue in detail. It seems feasible with additional markers of the dendrite and spines. But this is not developed in the manuscript. Also, it is not demonstrated to which extent this technique outperforms traditional methods for synaptic weight measurements like spine volume.

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  4. Reviewer #2 (Public Review):

    Over the last couple of decades, the development of fluorescent transgenic mouse lines (e.g thy1-GFP) and delivery techniques (e.g., in utero electroporation), as well as the democratization of recording methods in living animals (such as calcium imaging, high-density probes,) have strengthened the link between synaptic plasticity and behavior. Nevertheless, these methods are most of the time limited to hundreds of cells at best (very few in the case of patch-clamp recordings), or failed to achieve a clear synaptic resolution without affecting the tight equilibrium of endogenous proteins.

    In this paper, Graves, Roth, Tan, Zhu, Bygrave, Lopez-Ortega et al present an additional high-resolution optical tool to overcome these limitations. They generated a new knock-in mouse line that fluorescently labels all endogenous AMPA receptors (KI SEP-GluA1). In this mouse line, the extracellular N-terminal domain of the GluA1 subunit of AMPAR is tagged with super ecliptic pHluorin (SEP), a pH sensitive variant of GFP that fluoresces at neutral pH (at cell surface) and is quenched at acidic pH (within the cell). This tool thus avoids the use of antibodies or the over-expression of exogenous tagged receptors.

    They perform a set of convincing experiments showing that synaptic transmission, homeostatic and activity-dependent synaptic plasticity in vitro (Figs2-3), and behavior (Fig. 4), are not affected in KI SEP-GluA1 as compared to wild-type mice. Despite the obvious quality and viability of this mouse line (this is an important tool with no doubt), it is puzzling however that, while the level of GluA2 remains unchanged, the global expression of SEP-GluA1 is twice as low as the expression of GluA1 in wild-type mice (Fig.1). The manuscript would benefit from a clear brain-region specific comparison between the expression pattern of GluA1 and SEP-GluA1. At least, the author should discuss this point, and how this might affect the formation of GluA1/A2 heteromers, the dominant form of AMPAR in pyramidal neurons.

    Then, they provide strong evidence that SEP-GluA1 receptors are mobile in vivo (Fig. 6) and can thus accurately report synaptic plasticity, at least in anesthetized animals (Figs. 7-8). The authors make a point that "this novel SEP-GluA1 knockin mouse is the first tool that enables longitudinal tracking of synaptic plasticity underlying behavior at brain-wide scale with single-synapse resolution". However, given the high density of fluorescent synapses, it remains unclear how effective would be this mouse line in awake mice, and more specifically during behavior, in which movement artifacts could preclude the tracking and registration of the same population of SEP-GluA1 containing synapses over time.

    Finally, they present a new automated analytical tool to detect and register fluorescent synapses (Fig.7). Although the initiative is important and laudable (it is true that imaging approaches are usually plagued by the lack of user-friendly analysis tools), the method would benefit from a comparison with existing methods.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    Understanding the distribution of synaptic strength and plasticity in the brain is paramount for understanding neural circuit function underlying behavior. In the manuscript by Graves et al., the authors developed a novel mouse model for optical detection of synaptic strength and plasticity in live brains. Specifically, they modified the mouse genome by modifying the native GluA1 AMPAR subunit gene (gria1) with a pH-sensitive GFP (pHluorin) -tagged GluA1 at its N-terminus. This sensor is nearly maximally fluorescent when the pH is neutral and quenched in acidic environments and, therefore, preferentially marks AMPARs located on the plasma membrane. Since AMPARs are known to cluster in the postsynaptic density, AMPAR number is the predominant postsynaptic determinant of synaptic strength. Specifically, the trafficking of GluA1 AMPARs is responsible for LTP in CA1 hippocampus. The use of this novel genetic tool raises the possibility of monitoring synaptic strength optically, thus providing a strategy for massively parallel assessment of the distribution of synaptic strength in bulk brain tissue. Even more promising is the use of cranial windows and 2-photon microscopy to assess synaptic strength longitudinally, for example, during learning acquisition.

    The authors performed a comprehensive and rigorous set of control experiments using electrophysiology and behavior experiments. They demonstrated that modified GluA1 acts as native receptors and does not suffer from the shortcomings of overexpression approaches. The authors convincingly demonstrate that the modified receptors generate normal wild-type synaptic physiology and no behavioral alterations. Using glutamate uncaging, they showed that fluorescence changes at synapses were highly correlated with an electrophysiological assessment of synaptic strength and plasticity. Thus the data support claims that synaptic strength and plasticity could be assessed and monitored at unprecedented parallelization.

    Whether SEP-GluA1 can be used to quantify synaptic strength and its changes is uncertain due to an unknown ratio of GluA1/2 versus GluA2/3 receptors, the differential expression of GluA1 in different cell types, and the presence of GluA1-independent plasticities. Another potential shortcoming of the study is the lack of a ground truth demonstration of true synapses in vivo. Given the high-density of synapses, low z-resolution 2P microscopy (> 2 um), and the presence of a significant extrasynaptic pool, a confirmation of their results with superresolution or EM would be essential. Moreover, the lack of cell-type-specific labeling is likely to limit the tool's use for linking behavioral and microcircuit synaptic plasticity. It is possible that control experiments in acute brain slices could circumvent some shortcomings and provide a more quantitative workflow.

    Read the original source
    Was this evaluation helpful?