Establishment of transgenic fluorescent mice for labeling synapses and screening synaptogenic adhesion molecules
Curation statements for this article:-
Curated by eLife
eLife assessment
The fluorescently tagged SYT-1 mouse line will be useful for the field. Importantly, the authors used a comprehensive set of immunohistochemical and physiological experiments to demonstrate that the fluorescence tagging did not alter the function of SYT-1. These are important control experiments that will make the strain useful for physiological experiments in the future. However, the advance of this manuscript is less clear.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato ( Syt1- tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1- tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1- tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1- tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1- tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.
Article activity feed
-
-
Author Response
eLife Assessment:
The fluorescently tagged SYT-1 mouse line will be useful for the field. Importantly, the authors used a comprehensive set of immunohistochemical and physiological experiments to demonstrate that the fluorescence tagging did not alter the function of SYT-1. These are important control experiments that will make the strain useful for physiological experiments in the future. However, the advance of this manuscript is less clear.
We thank the editor for raising this point. In the revised manuscript, we performed additonal experiments including testing the expression level of Syt1-TDT and testing the co-labeling of Syt1-TDT with synaptic marker in situ. We also dicussed the advantage of our model compared with the existed ones in line 285 to 300 in the section of discusion. Briefly, we conclude the …
Author Response
eLife Assessment:
The fluorescently tagged SYT-1 mouse line will be useful for the field. Importantly, the authors used a comprehensive set of immunohistochemical and physiological experiments to demonstrate that the fluorescence tagging did not alter the function of SYT-1. These are important control experiments that will make the strain useful for physiological experiments in the future. However, the advance of this manuscript is less clear.
We thank the editor for raising this point. In the revised manuscript, we performed additonal experiments including testing the expression level of Syt1-TDT and testing the co-labeling of Syt1-TDT with synaptic marker in situ. We also dicussed the advantage of our model compared with the existed ones in line 285 to 300 in the section of discusion. Briefly, we conclude the advance of our models as follows: First, the Syt1-TDT could label synapse in situ, especially in glomerular layer of olfactory bulb (compared with B6SJL-Tg(Thy1-Syt1/ECFP)1Sud/J (Han et al. 2005)). Second, we provided a potential usage of our model in the study of electrophysiological recording and imaging in vivo, as the electrophyiological properties of neurons from Syt1-TDT mice are normal (not be analyzed in B6.Cg-Tg(Thy1-YFP/Syp)10Jrs/J and B6;CBA-Tg(Thy1-spH)21Vnmu/J (Umemori et al. 2004; Li et al. 2005)), which might be result from the relative low expression of Syt1-TDT compared with the native Syt1. Third, the neurons from the transgenic mice can be used in ASF screening by skiping the procedure of immunostaining. It will save the cost of time, reagents and work.
Reviewer #1 (Public Review):
In this manuscript, Zhang and colleagues created a transgenic mouse strain that expresses SYT-1-tdt in all neurons. They showed that the labelled SYT-1 colocalizes with multiple synaptic markers and label synapses in different regions. More importantly, they showed that the transgenic expression does not alter synaptic function using ephys assays. This is a straightforward paper that generated a useful reagent that will be used broadly.
We are grateful for the reviewer’s positive comments.
Reviewer #2 (Public Review):
Yang et al. produced a transgenic mouse line (Syt1-TDT) that could be used for labeling both excitatory and inhibitory synaptic sites in cultured neurons and in vivo neurons. The strength of the current study is to provide a series of thorough analyses to claim the applicability of this mouse line in the relevant neuroscience research field(s). The weakness is the potential impact/usefulness of this mouse line. To strengthen the merit of this mouse line, the authors should present evidence showing its advantage over other similar genetic approaches.
We thank the reviewer for raising this point. To strengthen the merit of this mouse line, we tested the application of Syt1-TDT in labeling synapse in situ. We found that the Syt1-TDT is highly overlapped with synapsin in the brain slice, especially in hippocampus, cerebellum and olfactory bulb, which suggest a potential usage of our model in imaging synapse in vivo. We also compared our transgenic model with the existed ones in line 285 to 300 in the section of discussion in the revised manuscript:
“Several fluorescently tagged synaptic protein transgenic mice model, such as YFP tagged synaptophysin and pHluorin tagged synaptobrevin have been developed to label synapses [49, 50]. While these models can label synapse well, it lacks the functional analysis of neurotransmitter release in the overexpressed neurons as synaptophysin and synaptobrevin were reported to play a role in regulating neurotransmitter release. Considering the overexpression of synaptobrevin or synaptophysin were reported to promote neurite elongation or enhance neurotransmitter secretion, the synaptic organization and synaptic transmission might be changed in these models. Weiping Han et al. in their previous work [47] have generated transgenic mice expressing a Syt1-ECFP fusion protein. The Syt1-ECFP mice expressed the fluorescent protein ECFP in the cortex, midbrain, and cerebellum. However, the expression pattern in their model showed some difference with ours: In the olfactory bulb, the Syt1-TDT signals were highly enriched in glomerular layer in our model, which was not observed in the previously reported Syt1-ECFP transgenic mice [47]. It suggested a potential application of our model in labeling synapse in glomerular layer of olfactory bulb compared with Syt1-ECFP transgenic mice.”
Reviewer #3 (Public Review):
Yang and colleagues provide a thorough characterization of a transgenic mouse model expressing fluorescently tagged synaptotagmin. In particular, they present key controls validating this mouse model as a tool, including co-localization of the tagged synaptotagmin with other synaptic markers as well as normalcy of synaptic transmission mediated by synaptic terminals expressing the tagged synaptotagmin. Importantly, the authors present data on the potential use of neuronal cultures obtained from these mice in synaptic co-culture assays. In these assays, synaptic cell adhesion molecules expressed on non-neuronal cell lines such as HEK-293 cells or COS cells are used to test the sufficiency of these molecules to trigger synapse assembly. This mouse model will be a useful addition to existing models expressing fluorescently-tagged synaptic vesicle proteins such as synaptophysin, synaptotagmin as well as synaptobrevin.
We are grateful for the reviewer’s positive comments.
-
eLife assessment
The fluorescently tagged SYT-1 mouse line will be useful for the field. Importantly, the authors used a comprehensive set of immunohistochemical and physiological experiments to demonstrate that the fluorescence tagging did not alter the function of SYT-1. These are important control experiments that will make the strain useful for physiological experiments in the future. However, the advance of this manuscript is less clear.
-
Reviewer #1 (Public Review):
In this manuscript, Zhang and colleagues created a transgenic mouse strain that expresses SYT-1-tdt in all neurons. They showed that the labelled SYT-1 colocalizes with multiple synaptic markers and label synapses in different regions. More importantly, they showed that the transgenic expression does not alter synaptic function using ephys assays. This is a straightforward paper that generated a useful reagent that will be used broadly.
-
Reviewer #2 (Public Review):
Yang et al. produced a transgenic mouse line (Syt1-TDT) that could be used for labeling both excitatory and inhibitory synaptic sites in cultured neurons and in vivo neurons. The strength of the current study is to provide a series of thorough analyses to claim the applicability of this mouse line in the relevant neuroscience research field(s). The weakness is the potential impact/usefulness of this mouse line. To strengthen the merit of this mouse line, the authors should present evidence showing its advantage over other similar genetic approaches.
-
Reviewer #3 (Public Review):
Yang and colleagues provide a thorough characterization of a transgenic mouse model expressing fluorescently tagged synaptotagmin. In particular, they present key controls validating this mouse model as a tool, including co-localization of the tagged synaptotagmin with other synaptic markers as well as normalcy of synaptic transmission mediated by synaptic terminals expressing the tagged synaptotagmin. Importantly, the authors present data on the potential use of neuronal cultures obtained from these mice in synaptic co-culture assays. In these assays, synaptic cell adhesion molecules expressed on non-neuronal cell lines such as HEK-293 cells or COS cells are used to test the sufficiency of these molecules to trigger synapse assembly. This mouse model will be a useful addition to existing models expressing …
Reviewer #3 (Public Review):
Yang and colleagues provide a thorough characterization of a transgenic mouse model expressing fluorescently tagged synaptotagmin. In particular, they present key controls validating this mouse model as a tool, including co-localization of the tagged synaptotagmin with other synaptic markers as well as normalcy of synaptic transmission mediated by synaptic terminals expressing the tagged synaptotagmin. Importantly, the authors present data on the potential use of neuronal cultures obtained from these mice in synaptic co-culture assays. In these assays, synaptic cell adhesion molecules expressed on non-neuronal cell lines such as HEK-293 cells or COS cells are used to test the sufficiency of these molecules to trigger synapse assembly. This mouse model will be a useful addition to existing models expressing fluorescently-tagged synaptic vesicle proteins such as synaptophysin, synaptotagmin as well as synaptobrevin.
-
