Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor

  1. Camille S Wang
  2. Natali L Chanaday
  3. Lisa M Monteggia
  4. Ege T Kavalali

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    Evaluation Summary:

    This paper studies spontaneous and evoked excitatory synaptic transmission in cultured hippocampal neurons using a genetically encoded fluorescent glutamate sensor. The central finding of this study is that after photobleaching, the spontaneous release of glutamate recovers rapidly, while the evoked release of glutamate recovers much more slowly. This study is potentially of very high interest to neurobiologists as there has been a long-running interest in understanding spontaneous versus evoked neurotransmitter release. Clarification of a few key technical issues central to the study is required to fully interpret the study.

    (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 #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

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  1. Version published to 10.7554/elife.76008 on eLife
  2. eLife

    Evaluation Summary:

    This paper studies spontaneous and evoked excitatory synaptic transmission in cultured hippocampal neurons using a genetically encoded fluorescent glutamate sensor. The central finding of this study is that after photobleaching, the spontaneous release of glutamate recovers rapidly, while the evoked release of glutamate recovers much more slowly. This study is potentially of very high interest to neurobiologists as there has been a long-running interest in understanding spontaneous versus evoked neurotransmitter release. Clarification of a few key technical issues central to the study is required to fully interpret the study.

    (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 #2 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The authors have taken a creative approach to addressing an important and controversial question, how much do the signaling pathways underlying spontaneous and evoked neurotransmission overlap? They have combined a number of approaches to examine small excitatory synapses from hippocampal neurons. In addition, this work focuses on the postsynaptic elements of synaptic transmission. The use of a fluorescent glutamate sensor is a major strength of the work as it allows localization of spontaneous and evoked release events to specific areas of the neuron unlike electrophysiological recordings where the synapse mediating a particular release event is usually unclear. Their experiments using fixed tissue and super resolution microscopy show that the fluorescent sensor accumulates near synapses. In the first half of the paper the authors validate the approach by examining how the likelihood of spontaneous and evoked release can be evaluated by this method and they compare the values with those obtained using electrophysiology under a range of conditions. A major finding is that the rate of recovery of fluorescence from the sensor, following photobleaching, appears to be very different for spontaneous and evoked events consistent with their thesis that the pathways reporting transmission are different for the two forms of release. However, their finding that stimulation did not impact the rate of photobleaching is puzzling and on first look appears inconsistent with the interpretation. Additional discussion of this point would enhance interpretation and increase its impact on the field. The work extends the study of synaptic function and allows localization of release to specific synapses or synaptic clusters.

  4. eLife

    Reviewer #2 (Public Review):

    This is a very interesting and well executed study from an established and very well respected group of synaptic neuroscientists. They have used iGluSnFR based glutamate imaging to examine the fundamental properties of spontaneous vs evoked neurotransmitter release. There are a number of exciting and intriguing findings in this study. First, the authors report that GluSnFR is enriched in synapses in culture. Second, they report that there is a significant portion of GluSnFR that is immobile in neurons. Finally, they show that after photobleaching, the detection of evoked glutamate release is very slow to recover while the detection of spontaneous glutamate release recovers much more quickly. These findings are exciting and impactful as great efforts have been devoted to understanding the mechanisms that differentially regulate evoked and spontaneous release of neurotransmitters. The findings also have important implications on how to use and interpret glutamate imaging modalities, which are gaining significant popularity. Overall this is an exciting and thought provoking study. There are a number of technical and conceptual issues with the study that should be clarified and currently limit the ability to interpret the findings with complete confidence.

    Strengths of the study:
    - Rigorous and quantitative analysis of spontaneous and evoked glutamate release.
    - Extensive validation of the imaging approach to answer the questions of interest.
    - Potentially very impactful finding of differential recovery of spontaneous and evoked glutamate release
    - Carefully analyzed and interpreted findings that shed new light on the spatial and temporal distinctions between spontaneous and evoked release.

    Weaknesses of the study:
    - Additional clarity is required to understand exactly how spontaneous events were detected and quantified.
    - There is a difference, potentially of impact on the findings of the study, between the area that was photobleached and the area in which recovery from photobleaching was studied. This needs to be clarified and its potential implications on the finding considered.

  5. eLife

    Reviewer #3 (Public Review):

    Wang CS et al investigate the localization of spontaneous and evoked vesicle fusion within a synapse to better understand a central question in neuronal communication. Given that spontaneous vesicle fusion is present in the brains of almost all organisms studied to date, far too little is understood about its role in the brain. The authors nicely document the use of a glutamate-sensitive fluorescent reporter (iGluSnFR to measure spontaneous and evoked vesicle fusion. Taking advantage of the iGluSnFR's rapid bleaching, the authors follow up previous molecular and physiological characterization of spontaneous vesicle fusion largely done in their labs with novel findings of unique spatial segregation of spontaneous vesicle fusion based on this bleaching characteristic. This manuscript has several high quality measurements and beautiful recordings made with excellent temporal resolution. Furthermore, the writing and presentation overall were very clear with interesting conclusions and discussion. While work from a number of labs has recently demonstrated restricted nano-domains for evoked vesicle fusion critical for our understanding of synaptic communication, unique sites for spontaneous release have not previously been reported postsynaptically. These findings suggest interesting future experiments to determine the unique receptors that localize (or not) at segregated sites of spontaneous vesicle fusion going forward. I was impressed by the use of the bleaching of a membrane probe to resolve the location of vesicle fusion. I have some questions and comments that I believe are important to be addressed to better understand the findings.

    1. The iGluSnFR probe has a very high affinity to provide it with the sensitivity for detecting single vesicle fusion. A concern would be if the sensor is also detecting vesicle fusion as spillover from adjacent boutons of untransfected neurons not directly forming a synapse with dendrites expressing GluSnFR.

    2. The difference in bleaching from glutamate perfusion compared to during rest or "no stimulation" for selectively impairing detection of evoked vs spontaneous release is striking and really interesting. That being said, it is very hard to understand the explanation the authors provide for the lack of difference between unstimulated and stimulated bleaching conditions if iGluSnFR bleaching requires glutamate release. Please provide more detail of the bleaching illumination intensity as well as changes in resting fluorescence of GluSnFR from the three bleaching conditions to better understand the interpretation.

  6. Version published to 10.1101/2021.12.10.472065v1 on bioRxiv