Visualizing synaptic dopamine efflux with a 2D composite nanofilm

  1. Chandima Bulumulla
  2. Andrew T Krasley
  3. Ben Cristofori-Armstrong
  4. William C Valinsky
  5. Deepika Walpita
  6. David Ackerman
  7. David E Clapham
  8. Abraham G Beyene

  • Curated by eLife

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    Evaluation Summary:

    This is a very exciting study that presents a novel approach to examining dopamine release with spatial precision that is so far unrivaled. This manuscript is also important and timely in the field of biosensor development and of potential interest to neuroscientists who study neurochemical release. It introduces a synthetic nanofilm with high spatiotemporal resolution and quantal sensitivity to dopamine measurement. By utilizing this technology to visualize sub-cellular dopamine efflux, the work provides new insights into the spatiotemporal dynamics and protein machinery of somatodendritic dopamine release. The authors identify hotspots for DA release and also provide evidence for DA release in the presence of TTX, suggesting the occurrence of quantal release.

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

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Abstract

Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.

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  1. Version published to 10.7554/elife.78773 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    (1) The authors mention that they did not observe DA release at sites that did not also have bassoon puncta. However, the data in Figures S13A, and B suggests that this statement may be true only to a rough approximation. If possible, the authors should verify this statement by quantifying the DopaFilm signals at bassoon positive and bassoon negative areas.

    We thank the reviewer for this important comment. The image in Figure S13A/B (now Figure 6—figure supplement 2A/B) was taken with a low magnification objective and we can see how it may paint a somewhat unconvincing picture of the localization between Bassoon and ∆F/F responses. However, because the specimen from which the data is collected is no longer available, we are unable to reimage and reanalyze this particular data.

    To strengthen our claim, we instead chose to offer an analysis that quantitatively examines the correlation between Bassoon expression and ∆F/F hotspots activity from a different dopamine neuron. In this analysis, we show that DopaFilm activity detected at a location is strongly correlated with Bassoon expression at the same location. This new analysis is consistent with our observation that Bassoon plays an important role in orchestrating release in dendritic processes.

    Changes to manuscript in response to this comment:

    (1) A new Figure 6—figure supplement 1 is added. (2) Accompanying text in manuscript referencing this new figure and updates to Methods section describing how this analysis was carried out.

    We modified the original sentence that read:

    “Importantly, while we observed that presence of Bassoon did not necessarily indicate presence of DopaFilm activity, we did not observe DopaFilm activity from a dendritic process that did not have Bassoon puncta (Figure S13).”

    The sentence now reads:

    “Importantly, density of Bassoon expression at a location correlated positively with the magnitude of ∆F/F activity measured by DopaFilm at the same location (Figure 6— figure supplement 1-2).”

    (2.1) In Fig 7C, the synaptobrevin2 staining does not seem to overlap well with the MAP2 or TH-GFP staining. Please comment on whether the synaptobrevin staining shown here represents staining in neighboring glutamatergic cells that are present in the co-cultures.

    The non-overlapping synaptobrevin-2 signal shown in Figure 7 are from other neurons in culture. We have clarified this in the text.

    Changes to manuscript in response to this comment:

    (1) We modified the figure caption for Figure 7 with this additional sentence:

    “Red puncta that do not colocalize with TH-GFP are from non-dopaminergic neurons in the co-culture system.”

    (2.2) Related to this, did the authors find any dependence of dopamine release here on glutamatergic transmission from cortical neurons? Please comment on this.

    We would first like to thank the reviewer for encouraging us to examine the effect that glutamatergic transmission may have on dopamine release in the co-culture system. To investigate this, we carried out experiments in which AMPA-type glutamate receptor antagonists NBQX and NMDA-type glutamate receptor antagonist D-AP5 were bath applied to the culture system while imaging release from dopamine neurons. We first examined neurons from which DopaFilm activity can be detected from spontaneous spiking events (that is, in which we applied no external stimulus to generate activity but from which we measured action potential driven, synchronous release). We imaged from these neurons under ACSF (our normal imaging buffer) and then applied NBQX (10 µM). DopaFilm activities that were detected before application of NBQX were absent in the post drug imaging sessions (see Figure 3—figure supplement 2A-B). Application of NBQX was sufficient to abolish the activity. We observed this phenomenon from n = 4 dopamine neurons.

    Additionally, we examined the extent to which glutamatergic currents contributed to dopamine neuron depolarization during evoked activity imaging. To investigate this, we carried out evoked imaging before and after glutamate receptor blockade with a combined application of NBQX and D-AP5. Here, such treatment resulted in reduced DopaFilm activity as measured by the peak amplitude of ∆F/F traces and the area under the curve (AUC) of ∆F/F traces (Figure 3—figure supplement 2C-E).

    Glutamatergic regulation of dopamine release is an interesting phenomenon that is worthy of a systematic exploration in an independent study. There are several outstanding questions and controversies in this field that are beyond the scope of the current study. However, these preliminary data suggest to us that our system may be suitable for examining how glutamatergic transmission regulates dopamine release, and if such regulation can occur independent of dopamine cell body firing. Our ability to measure dopamine release with synaptic spatial resolution may offer new insights into these phenomena in future studies.

    In light of these new experiments, we have made the following changes to the manuscript.

    Changes to manuscript in response to this comment:

    (1) A new supplementary figure (Figure 3—figure supplement 2) that shows the effect of glutamate transmission on DopaFilm activity.

    (2) A new paragraph in manuscript discussing these results. The new paragraph reads:

    “In our study, dopamine neurons are co-cultured with cortico-hippocampal neurons, and we explored if glutamatergic activity from neurons in co-culture could influence dopamine release. To investigate this, we carried out experiments in which AMPA-type glutamate receptor antagonist NBQX and NMDA-type glutamate receptor antagonist D-AP5 were bath applied to the co-culture system while imaging release from dopamine neurons. We first examined neurons from which DopaFilm activity can be detected from spontaneous spiking events in which we applied no external stimulus to generate activity. We imaged from these neurons under artificial cerebrospinal fluid (ACSF, our normal imaging buffer) and then bath applied NBQX (10 µM). DopaFilm activities that were detected before application of NBQX were absent in the post drug imaging sessions (Figure 3—figure supplement 2A-B). Application of NBQX was sufficient to abolish these activities. Additionally, we examined the extent to which glutamatergic currents contribute to dopamine neuron depolarization during evoked activity imaging. To investigate this, we carried out imaging before and after glutamate receptor blockade with a combined application of NBQX and D-AP5. Here, such treatment resulted in reduced dopamine release as measured by the peak amplitude of ∆F/F traces and the area under the curve (AUC) of ∆F/F traces (Figure 3—figure supplement 2C-E). In sum, these results indicate that DopaFilm offers an opportunity for direct measurement of dopamine release under pharmacological perturbations and suggests that our in vitro culture system may permit simplified explorations of local chemical circuitries that control dopamine release in the absence of complex circuit effects that may be encountered in vivo.”

    (3) In Figure 4, optical stimulation results in DA release and fluorescence increases at multiple hotspots. Interestingly, the change in fluorescence reaches very similar amplitudes across hotspots for each stimulation (compare dF/F at first red symbol across hotspots for example). Does this indicate saturation of the nanosensor? Interestingly, this seems to be true for the third stimulus as well, after depression when the signals are much smaller. By contrast in the dendrites, this doesn't seem to be the case, as shown in Fig S12. Better clarification on this point will inform whether DopaFilm can be used to probe synaptic release properties such as variance, etc. Please comment on this.

    The reason the traces appear to have the same peak amplitude is because of the scale of the figure. A closer look at the traces, when drawn on the same y-axis, shows that there is variability in the peak amplitude achieved across ROIs. To visualize the ∆F/F traces where one can better appreciate the variabilities observed from one hotspot to the next, we have created a new supplementary figure (Figure 4—figure supplement 1) that addresses this comment as well as several other comments. Additionally, we would like to point out that other data presented in this study show that there is variability in the measured peak ∆F/F values and sensor saturation is not observed (Figure 2B, Figure 3I, Figure 4E). However, it is possible, under some cases of high dopamine release, that we may observe a non-linear response from DopaFilm.

    Changes to manuscript in response to this comment:

    (1) We generated a new supplementary figure, Figure 4—figure supplement 1C-D, to go along with main Figure 4 to help improve the presentation and visualization of the data. Figure 4—figure supplement 1 has several panels that addresses this comment and several others from the reviewer.

    (4) Short-term plasticity. The authors suggest that dopamine neurons can sustain robust levels of release with no depletion but do not directly show this. Please provide time courses of DA release both from axons and dendrites during repeated stimulation. Relatedly, data shown in Fig 3I shows multiple stimulations without indicating the interstimulus interval. Please report the interstimulus interval for these experiments. The text mentions 1 stim per 2-3 min, but this is unclear. Lastly, optical stimulations in Figure 4C demonstrate multiple stimulations over time. It would be useful to see this quantified/normalized to the first stimulation.

    We would like to thank the reviewer for encouraging us to clarify some of our presentation of the data. Figure 3A was meant to provide the reader with evidence for robustness of response across stimuli but we concede that more can be done to support our claims in quantitative terms. Accordingly, we have now provided time course of response from DopaFilm hotspots collected from an extended imaging session. We did this for axons and dendrites in two separate supplementary figures (Figure 3—figure supplement 1A/B for axons, Figure 5—figure supplement 4 A-B for dendrites). In addition, we have clarified the interstimulus intervals both in the body of the text as well as figure captions. For data in Figure 3, the time on the x-axis provides time interval between stimuli. These are multiple stimuli delivered within the same imaging session, and the short-term depression observed typically recovers after a rest period.

    Changes to manuscript in response to this comment:

    (1) We generated figure panels A and B in Figure 3—figure supplement 1 that will serve as a supplement for Figure 3. We produced a similar data for dendrites in Figure 5— figure supplement 4 A-B.

    (2) We clarify stimulation and imaging protocols in the accompanying figure captions.

    (5) Kinetics of release measured with DopaFilm. Figures 2B and 2D suggest that dendritic release is fast but the scaling of the traces shown makes it difficult to see onset timing. Please provide measurements of the averaged time-to-peak for both axonal DA release and somatodendritic release. Also, it would be helpful to the reader to discuss how these times compare to the time course of DA release as measured using dLight or GRABDA, carbon fibers and D2 IPSCs.

    Thank you for this comment. We appreciate that the discussion of the turn-on and clearance kinetics in axons and dendrites, and how these compare with other methods of measuring dopamine release, will be of interest to the broader readership. Accordingly, we now provide a comparison of time-to-peak (τpeak) and first order decay time constant (τoff) for activities measured in axons and dendrites. We discuss these values in the context of values reported for other tools.

    Changes to manuscript in response to this comment:

    (1) A new panel in supplementary Figure 2—figure supplement 1 is generated that provides information on the on and off temporal properties.

    (2) A discussion of these values is provided in the results section of the main text. The new text reads:

    “The turn-on and clearance kinetics of the measured transients in axons were 0.46 ± 0.16 s (Mean ± SD) for time-to-peak (τpeak) and 3.83 ± 0.8 s (Mean ± SD) for first order decay time constant (τoff) (Figure 2—figure supplement 1). The turn on kinetics is slower than those reported for the genetically encoded dopamine sensors GRABDA (≈100 ms) and dLight (reported as τ1/2 of ≈10 ms followed by a plateau of ≈100 ms).32,33 On the other hand, decay kinetics appears to be slower than dLight (reported as τ1/2 ≈ 100 ms) and comparable to or faster than those reported for GRABDA ( ≈ 3 – 17 s for variants). For comparison, GIRK-current based dopamine dynamics measurements exhibited τpeak ≈ 250 ms whereas carbon fiber recordings peaked in τpeak ≈ 300 ms.34 This suggests that the kinetic properties of DopaFilm transients are comparable with the range of reported values from existing tools.”

    Reviewer #3 (Public Review):

    Bassoon spots with no dopamine release. Do these silent sites always remain silent? What is the percentage of these 'silent' sites compared to all observed dopamine release events?

    We would like to thank the reviewer for encouraging us to compute the fraction of boutons that are release-competent in a given axonal arbor. We found that this value varied greatly, with some FOVs producing up to 65% release-capable boutons, whereas others had just small fraction (5%) that participated in release.

    Changes to manuscript in response to this comment:

    (1) We added the following sentence to the results section at the relevant location:

    “The percentage of release-competent boutons varied greatly in axonal arbors, ranging from 5% in some FOVs to 65% in others, with a mean of 32% of putative boutons participating in release.”

  3. eLife

    Evaluation Summary:

    This is a very exciting study that presents a novel approach to examining dopamine release with spatial precision that is so far unrivaled. This manuscript is also important and timely in the field of biosensor development and of potential interest to neuroscientists who study neurochemical release. It introduces a synthetic nanofilm with high spatiotemporal resolution and quantal sensitivity to dopamine measurement. By utilizing this technology to visualize sub-cellular dopamine efflux, the work provides new insights into the spatiotemporal dynamics and protein machinery of somatodendritic dopamine release. The authors identify hotspots for DA release and also provide evidence for DA release in the presence of TTX, suggesting the occurrence of quantal release.

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

  4. eLife

    Reviewer #1 (Public Review):

    Chamdima Bulumulla et al. report a synthetic 2D-composite nanofilm to visualize the dopamine transmission with sub-cellular resolution, favored temporal properties and quantal sensitivity. This work reveals that the somatodendritic dopamine release originates from the dendrites, including major dendrites, fine dendritic processes and dendritic arbors, but not soma. The dynamics of dendritic release are similar to those of axonal release but with a restricted spatial extent at the release site. By combining post hoc immunofluorescent super-resolution imaging, the authors find dendritic hotspots are enriched with presynaptic active zone protein Bassoon and co-localized with the SNARE complex protein, VAMP2. These data provide new insights into the spatiotemporal dynamics of somatodendritic dopamine release and also cast light on the potential molecular machinery.

  5. eLife

    Reviewer #2 (Public Review):

    In this study, Bulumulla and colleagues explore the use of near-infrared dopamine nanosensors to visualize dopamine release from axons and dendrites. In the original description of this sensor published by the last author, the dopamine nanosensor was used to examine bulk dopamine release in tissue slices. Here, the authors take an innovative approach by applying a thin coat of the nanosensor to coverslips (named DopaFilm). This enables them to image dopamine release from cultured dopaminergic neurons with much higher spatiotemporal resolution.

    This is a very exciting study that presents a novel approach to examining dopamine release with spatial precision that is so far unrivaled. The authors identify hotspots for DA release and also provide evidence for DA release in the presence of TTX, suggesting that they can image quantal release. In addition, the results here present the first clear visualization of somatodendritic dopamine release to date. Importantly, whether somatodendritic release genuinely occurs is still a matter of debate - some in the field believe that axonal fibers are the main source of DA released in midbrain VTA and SNc. Therefore, this manuscript presents an excellent study that contributes significantly to our understanding of dopamine release.

    Comments:

    The authors mention that they did not observe DA release at sites that did not also have bassoon puncta. However, the data in Figures S13A, and B suggests that this statement may be true only to a rough approximation. If possible, the authors should verify this statement by quantifying the DopaFilm signals at bassoon positive and bassoon negative areas.

    In Fig 7C, the synaptobrevin2 staining does not seem to overlap well with the MAP2 or TH-GFP staining. Please comment on whether the synaptobrevin staining shown here represents staining in neighboring glutamatergic cells that are present in the co-cultures.
    Related to this, did the authors find any dependence of dopamine release here on glutamatergic transmission from cortical neurons? Please comment on this.

    In Figure 4, optical stimulation results in DA release and fluorescence increases at multiple hotspots. Interestingly, the change in fluorescence reaches very similar amplitudes across hotspots for each stimulation (compare dF/F at first red symbol across hotspots for example). Does this indicate saturation of the nanosensor? Interestingly, this seems to be true for the third stimulus as well, after depression when the signals are much smaller. By contrast in the dendrites, this doesn't seem to be the case, as shown in Fig S12. Better clarification on this point will inform whether DopaFilm can be used to probe synaptic release properties such as variance, etc. Please comment on this.

    Short-term plasticity. The authors suggest that dopamine neurons can sustain robust levels of release with no depletion but do not directly show this. Please provide time courses of DA release both from axons and dendrites during repeated stimulation. Relatedly, data shown in Fig 3I shows multiple stimulations without indicating the interstimulus interval. Please report the interstimulus interval for these experiments. The text mentions 1 stim per 2-3 min, but this is unclear. Lastly, optical stimulations in Figure 4C demonstrate multiple stimulations over time. It would be useful to see this quantified/normalized to the first stimulation.

    Kinetics of release measured with DopaFilm. Figures 2B and 2D suggest that dendritic release is fast but the scaling of the traces shown makes it difficult to see onset timing. Please provide measurements of the averaged time-to-peak for both axonal DA release and somatodendritic release. Also, it would be helpful to the reader to discuss how these times compare to the time course of DA release as measured using dLight or GRABDA, carbon fibers and D2 IPSCs.

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript reports a new technology achievement of directly visualizing dopamine release from single synapses and with millisecond time resolution. Dopamine release is usually detected by electrochemical or microanalysis assays, which does not provide spatial or temporal information. Here, the authors utilized a recently developed dopamine nanosensor based on oligonucleotide-functionalized single-wall carbon nanotubes. DopaFilm is a thin layer of these nanosensors whose infrared fluorescence is highly sensitive to dopamine. When dopaminergic neurons were cultured on DopaFilms, local dopamine released from axons, dendrites, or varicosities can be reliably detected by fluorescence imaging of the DopaFilm. This manuscript includes extensive data to support the claim, which is further strengthened by post hoc immunostaining of proteins that are specific to dopaminergic neurons, dendrites, or synapses.

    Overall, this is a high-quality work supported by extensive data. The engineering of the nanosensor has been previously reported and has been validated by different research groups. This work makes the nanosensors into two-dimensional DopaFilm to resolve signals from individual synapses. This manuscript shows that DopaFilm affords excellent sensitivity for detecting real-time dopamine release from individual varicosities or synapses. The spatiotemporal dynamics of dopamine release in the dendritic processes of dopaminergic neurons is a poorly understood phenomenon. Therefore, technologies reported in this work would be of interest to future studies of the underlying mechanisms of this process. The conclusions of this paper are well supported by data, but some questions would need to be clarified. For example, the authors showed some Bassoon spots with no dopamine release. Do these silent sites always remain silent? What is the percentage of these 'silent' sites compared to all observed dopamine release events?

  7. Version published to 10.1101/2022.01.19.476937v1 on bioRxiv