Coordinated dynamics of excitatory and inhibitory synapse assembly

Reviewer #1 (Public review):

Summary:

By imaging the dynamics of synaptic proteins in cultured neurons, this study presents significant findings regarding the dynamics of excitatory and inhibitory synaptic proteins during development. The evidence shows that the ratios of excitatory and inhibitory synaptic proteins are stable during synapse development. This discovery advances our understanding of the complex mechanisms governing synapse formation. The strength of the evidence is robust, as it is supported by a combination of biological assays and endogenous labeling.

Strengths:

This research sheds light on the dynamics of the excitatory and inhibitory synapses during development. It is crucial to understand that while excitatory synapses and inhibitory synapses are developed independently, the ratio of their number is relatively stable during development, maintaining a stable excitatory/inhibitory ratio.

Important findings and implications in the research include:

(1) Persistent Synapse Dynamics: Excitatory and inhibitory synapses remain highly dynamic even in mature neurons (DIV12-14), challenging the dogma that synaptic structures are stable after the synaptogenesis stage.

(2) Maintained E/I Balance: Despite ongoing synapse turnover (formation/elimination) and presynaptic terminal reduction, the overall density and ratio of excitatory-to-inhibitory synapses remain relatively stable during circuit maturation (Figure 7).

(3) Developmental Shifts: While presynaptic compartments decrease over time, postsynaptic sites increase, suggesting independent regulation of pre- and postsynaptic elements within a stable E/I framework.

Weaknesses:

This study focuses on specific synaptic proteins within synapses, which may not fully represent the dynamics of other synaptic machinery; also, whether similar observations exist in vivo is still unknown. Further research is needed to explore the implications of these findings in more complex neuronal environments.

Reviewer #2 (Public review):

Summary:

The Garbett et al. identified a critical need to begin to understand the interplay between the assembly, maturation, and elimination of excitatory and inhibitory synapses. They also detail the lack of reliable tools to address this gap in knowledge. Here, the authors developed synaptic reporters expressed by lentiviruses (mClover3-Homer1c, HaloTag-Syb2, and tdTomato-Gephyrin). They combined these reporters with resonance scanning confocal imaging to measure synapses over a 15-hour period during neuron development and in mature neurons in primary hippocampal cultures. Using these reporters in the same neuron, the authors compared the ratios of postsynaptic excitatory and inhibitory specializations that co-localize with presynaptic terminals during development and in mature neurons and found that they are stable across time points. Finally, the authors developed CRISPR/Cas9 tools (TKIT) to knock-in endogenous fluorescent tags (GFP/tdTomato-Gephyrin) or epitope tags (HA-Bassoon and HA-Homer1) to begin to study synapse dynamics using endogenous proteins. I believe this paper highlights an important gap in knowledge and begins to offer methodologies to determine the dynamic coordination between excitatory and inhibitory synapses.

Strengths:

(1) The experiments are well-designed and carefully controlled.

(2) The authors carefully validated the reporter and TKIT constructs.

(3) The authors provide strong proof-of-principle for the use of the reporter constructs to track synapse formation, maintenance, and elimination over a 15-hour period.

(4) Ingenious use of technologies (reporters, TKIT, and resonance scanning confocal microscopy) to develop a platform for future studies of synapse dynamics.

(5) Strong evidence supporting that the ratio of excitatory and inhibitory synapses (those that oppose syb2) stays constant through development.

Weaknesses:

Overall, this is a well-executed study that develops tools to simultaneously image excitatory and inhibitory synapse dynamics and represents an important first step to address the fundamental question regarding the coordination between these two types of synapses.

Minor weaknesses of the manuscript include:

(1) The lack of a characterization of endogenous Homer1-positive excitatory synapses using TKIT.

(2) Discussion about other approaches to study excitatory and inhibitory synapses using endogenous proteins (e.g., intrabodies - FingR or nanobodies) should be included.

(3) The activity state of a neuron and/or a synapse might alter the dynamic properties (formation, maintenance, and/or elimination). A discussion on whether the overexpression of Homer1 and/or gephyrin might alter synapse/neuron activity would provide greater interpretability of the results. A discussion of the potential limitations and benefits of the reporter and TKIT approaches would be beneficial.

(4) A description and interpretation of the computational approach to calculate particle tracking would be helpful. I found that particle tracking figures, while elegant, are difficult to interpret.

Reviewer #3 (Public review):

In the present study, the authors describe the development of new tools and imaging strategies to assess the concomitant development of excitatory and inhibitory synapses in dissociated neuron cultures. To this end, they generate fluorescently tagged constructs of excitatory and inhibitory synapse marker proteins using either conventional overexpression or CRISPR-based strategies. They then image these marker proteins over a timespan of 15 hours to assess synaptic dynamics at different developmental timepoints. Based on their data, they conclude that excitatory and inhibitory synapse development occur in concert to maintain a functional balance despite individual synapse turnover.

Overall, this study addresses an interesting question, i.e., the interplay between the development of excitatory and inhibitory synapses, which has important implications, particularly for neurodevelopmental disorders in which the balance of excitation and inhibition is disrupted. The experiments are technically solid and well-executed, and the individual images are highly compelling.

However, a number of aspects remain to be addressed in order for the study to support the claims made by the authors. First, the novelty aspect of the development of the fluorescently tagged synaptic proteins is unclear, since reporters of this nature are in routine use in many labs. Second, the analysis of the acquired images often seems incomplete, with only example images but no quantification shown, or the distinction between spatial and temporal dynamics appearing unclear. Third, given this incomplete analysis, the interpretations of the authors are not always convincingly supported by the data presented. In conclusion, substantial improvements are required to render the main messages of the study clear and compelling.