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.

Figures and data

Characterization of live imaging reporters to simultaneously monitor presynaptic and excitatory postsynaptic compartments

Visualization of distinct populations of excitatory synapses

Excitatory synapse tracking in both developing and mature hippocampal neurons

Live imaging the dynamics of inhibitory synapses

Characterization of TKIT CRISPR/Cas9-based reporters to label endogenous pre- or postsynaptic compartments

Long-term imaging of endogenous inhibitory postsynaptic Gephyrin

Simultaneous visualization of excitatory and inhibitory synapse dynamics during synaptogenesis

CRISPR KI sequences, AAV and Lenti titering primers, and primers for estimating the tagging efficiency of the TKIT method.