The synapsin-dependent vesicle cluster is crucial for presynaptic plasticity at a glutamatergic synapse in male mice

Abstract

Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity - i.e. decreased facilitation and post-tetanic potentiation - but increased long-term potentiation in male synapsin triple knockout mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from knockout animals. Our results indicate that all synapsin isoforms, including synapsin III, are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse.

Significance statement

Synapsins cluster vesicles at presynaptic terminals and shape presynaptic plasticity at giant hippocampal mossy fiber boutons . Deletion of all synapsin isoforms results in decreased short- but increased long-term plasticity.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8376891.

This review reflects comments and contributions from Alberto J. Gonzalez-Hernandez, Ryan Cubero & Femi Arogundade. Review synthesized by Ryan Cubero.

The study builds up on a previous study (Owe et al., 2009) that observed impaired frequency facilitation and reduced distal vesicle density in symptomatic synapsin I/II double knockout mice, and fills the gap by unraveling the role of synapsins in the presynaptic mediated plasticity by evaluating the effects of the total loss of synapsins using a triple KO for synapsin I, II and III (SynTKO) in a region of the brain which expresses synapsin III isoform in adult male mice (hippocampal mossy fibers - CA3 glutamatergic synapses). The study follows rigorous ethical and methodological guidelines, utilizing various experimental techniques, including electrophysiological recordings and transmission electron microscopy, to comprehensively analyze synaptic properties in synapsin triple knockout mice. The study reveals that the absence of all synapsin isoforms leads to altered excitability, vesicle organization, and plasticity in mossy fiber boutons, providing insights into the critical role of synapsins in synaptic function and plasticity.

Positive aspects of the paper:

The findings offer valuable insights into how the absence of all synapsin isoforms affects synaptic properties, potentially advancing our knowledge of neurological conditions related to synaptic dysfunction.
The study employs a well-structured study design, utilizing two age groups (presymptomatic and symptomatic) and comparing C57BL/6J control mice with SynTKO mice. This design allows for the investigation of changes before and after the onset of epileptic seizures in SynTKO animals.
The study rigorously examines synaptic function and structure, employing various assays and statistical analyses. This scientific rigor strengthens the validity of the results.
The use of blinding during data analysis is a positive aspect of the study. The experimenter was blinded to the treatment of slices, ensuring that the analysis is objective and not influenced by prior knowledge.
The authors reported the study in accordance with the SAGER guidelines and ARRIVE guidelines 2.0. Such practices should be recognized and highly commended.

Aspects that need to be addressed:

The measurements of Paired Pulse Ratio need to be improved. In Figure 1-2 panels b and c, the SynTKO condition has a replicate out of the range (which could be a potential outlier). This can be potentially addressed by increasing the number of replicates to really depict a better representation of the data dispersion. Furthermore, for panel c, the representative traces should be replaced to ones that represent the median. Ideally, the point of the condition selected for the trace can be highlighted in the graph (e.g. clear fill and same color outline).
In Figure 3c, the authors find a peculiar normalized fEPSP dynamics in SynTKO mice where the fEPSP slowly reaches a peak and slowly decreases, compared to control mice where the peak is reached immediately and decreases abruptly. Perhaps, the authors can discuss what could cause this altered LTP maintenance, given that there is reduced facilitation and PTP, but unimpaired paired-pulse ratio.

Minor comments:

In Figure 1c, the related text says "In SynTKO the amplitudes reached a plateau after 15 stimuli, whereas in WT animals, the amplitudes increased until the end of the 1 Hz stimulation". However, in the plot, there is a plateau for the WT condition around 20-25 stimuli. This sentence needs to be clarified or omitted if the data did not clearly depict this continuous increase.
As a general but minor comment, the representative traces of individual experiments should represent points close to the median (or average) and the point where the trace comes from can be highlighted in the plot.
The TEM images seem to show altered structural abnormalities in mossy fiber boutons in SynTKO mice compared to controls. Were there observed differences in synaptic bouton density?

Comments on reporting:

The statistical analyses are reported correctly in the methods section and table 5. Excluded recordings were also properly justified and detailed in table 6.

Suggestions for future studies:

In this mossy fiber-CA3 synapse, which has been reported to be highly plastic, it would be interesting to also explore what happens in the SynTKO model in presynaptic long term depression (caused by a low frequency stimulation protocol).
Inspecting potential differences in glutamate receptors in mossy fiber presynaptic boutons between SynTKO and control mice could help elucidate the observed differences in synaptic physiology.
Future experiments aiming to understand the role of synapsins (or the lack thereof) in inhibitory neurons will give us a clearer picture of the role of synapsins in synaptic function. It will also be interesting to explore how network connectivity is altered due to total loss of synapsins.

Competing interests

The author declares that they have no competing interests.

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The synapsin-dependent vesicle cluster is crucial for presynaptic plasticity at a glutamatergic synapse in male mice

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