Optical analysis of the action range of glutamate in the neuropil
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Evaluation Summary:
The authors address the spatial spread of glutamate outside of synapses, with the surprising conclusion that glutamate released at one synapse can strongly activate receptors at neighboring synapses. This manuscript should interest those studying neural signaling and techniques associated with that field. However, caveats of the advanced techniques used to address this difficult question limit the strength of the main conclusion.
(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. The reviewers remained anonymous to the authors.)
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Abstract
The wiring scheme of neurons is key to the function of the brain. Neurons are structurally wired by synapses and it is a long-held view that most synapses in the CNS are sufficiently isolated to avoid cross-talk to AMPA receptors of neighboring synapses. Here we report in hippocampal brain slices that quantal glutamate release activated optical reporter proteins >1.5 µm distant to the releasing synapse. 2P-glutamate uncaging was used to quantitatively probe glutamate spread in the neuropil. Releasing ∼35000 molecules of glutamate (∼5 vesicles) at a distance of 500 nm to a spine generated an uncaging EPSC reaching ∼30% of the quantal amplitude at synaptic AMPA-Rs. The same stimulus activated ∼70% of the quantal amplitude at NMDA-Rs and still generated clear current and calcium responses when applied at >= 2 µm remote to the spine. Extracellular spread of glutamate on the sub-micrometer scale appeared cooperative and caused supra-additive activation of AMPA-Rs in a spine. These observations are not predicted by previously used models of glutamate diffusion in the neuropil. An extracellular glutamate scavenger system weakly reduced field potential responses but not the quantal amplitude, indicating that a cross-talk component regularly contributes to synaptic transmission. Our data suggest that slight synaptic crosstalk responses at AMPA receptors of ∼2-4 adjacent synapses may be common (>70 synapses for NMDA receptors). Such broadcasting of synaptic signals to very local neighborhoods could stabilize network learning performance and allow for integration of synaptic activity within the extracellular space.
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Author Response:
Reviewer #1:
This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the …
Author Response:
Reviewer #1:
This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the interpretations reported here. However, I believe the authors have failed to take into account significant technical limitations inherent in the technologies they apply. These include spatial averaging of fluorescence, possible saturation of iGluSnFR and diffusive exchange of (caged) glutamate during uncaging. As a result, the conclusion is wholly unproven. Indeed, I believe it highly probable that all of the data in favor of distal activation will prove to be consistent with synapse specificity and the presence of technical artifacts related to spatial averaging of fluorescence signals and diffusive exchange of (caged) glutamate during uncaging.
We agree that there are technical limitations and that the interpreration of signals recorded from near synapses is difficult. This concerns the length constants we describe and name SARGe. Our usage of those terms in the results may have suggested we propose the value of lambda istelf well dscribes the action range of glutamate. This is not the true as the reviewer states and in the beginning of the discussion section we note this limitation.
However, our interpretation that glutamate may regularly activate AMPA-R in neighboring synapses is not based on lambda values (see discussion).
It is based on the facts that a) ~5% iGluSnFr responses are observed at more than 1.5 µm remote to a synapse and b) uncaging at 500 nm produces a current response of ~38% of the quantal synaptic amplitude. Here, the remarks of the reviewer are incorrect: a) is not affected by volume averaging or saturation of iGluSnfr and previous models predict an activation of upto 1-2% only. We have shown this by simulation in an appeal letter which unfortunately was not forwarded to the reviewer. b) is not increased by “diffusive exchange of glutamate during uncaging”. In fact, releasing the same amount glutamate for a longer period reduces distant receptor activation and current models predict an 2-4 fold lower activation of AMPA-R than we observe here. This was also shown by simulation in the appeal letter but a further exchange with the reviewer on this was not permitted by the editors.
Reviewer #2:
Matthews, Sun, McMahon et al. addresses the extent of the spread of the neurotransmitter glutamate into the extracellular space. The authors use a combination of imaging techniques, 2-photon glutamate uncaging and electrophysiology to conclude that vesicular glutamate release reaches nearby, adjacent synapses. Although this is an interesting question, and one that has been addressed many times previously, I have several technical concerns about the strength of the conclusions that reduces my enthusiasm.
Unfortunately, only this general part of comments of reviewer 2 is published so that we cannot meaningfully rule out/comment on the reviewer’s concerns.
Reviewer #3:
This is an interesting paper combining several impressive techniques to argue that synaptically released glutamate is allowed to diffuse to and activate receptors at much greater distance than previously thought. iGluSnFR recordings show that glutamate released from single vesicles activates the indicator with a spatial spread (length constant) of 1.2 um, substantially farther than previous estimates based on the time course of glutamate clearance by glial transporters (PMC6725141). Similar parameters are observed with spontaneous and evoked events, large or small, or when glutamate is released via 2P uncaging. Further uncaging experiments show that both AMPARs and especially NMDARs are activated a substantial distance. AMPARs, previously thought to be recruited only within active synapses, are activated with a spatial length constant that compares quite closely with the average distance between synapses in the hippocampus. More heroic experiments and some geometric calculations show that this behavior enables neighboring synapses to interact supralinearly. The results suggest that "crosstalk" between neighboring synapses may be substantially more common than previously thought.
The experiments in this paper appear carefully performed and are analyzed thoroughly. Despite all of the quantitative rigor and careful thought, however, the authors fail to reconcile convincingly their results with what we know about neuropil structure and the laws of diffusion. There are very good data in the literature regarding the extracellular volume fraction and geometric tortuosity of the neuropil, the diffusion characteristics of glutamate and the time course of glutamate uptake. These data more or less demand that synaptically released glutamate is diluted over a much smaller spatial range than that suggested here. In the Discussion, the authors suggest that this discrepancy might reflect a simplified view of the neuropil as an isotropic diffusion medium (PMC6763864, PMC6792642, PMC6725141), whereas a more realistic network of sheets and tunnels (PMC3540825) might prolong the extracellular lifetime of neurotransmitter. I like this idea in principle, but there is no quantitative support in the paper for the claim - in fact, it seems at odds with the authors' very nice demonstration that diffusion appears to be similar in all directions (Figure 3B). I don't necessarily think a solution is within the scope of this single paper, but I would suggest that the authors acknowledge the present lack of a compelling explanation.
Our results are not predicted by the modelling studies cited that is correct and this makes them important in our eyes. But it is important to note that those modelling/simulation studies use a strong simplification and view the extracellular space/ the neuropil as a porous medium. This is a powerful approach but it is only a valid description when considering diffusion distances of several micrometer - it is not applicable on the sub micron scale of neighboring synapses (PMID: 15345540 p1608; PMID: 7338810 p227, and DOI: 10.1088/0034-4885/64/7/202). This drawback of the simulation has been overlooked and the reviewer seems not to be aware of it and we point this out at the end of the discussion section. We do not suggest anisotropy near a synapse nor a particular perisynaptic geometry such that there would be specific channels from one synapse to the next; we don’t, we also assume that the neuropil is random (as shown by PMID 9547224) - instead everywhere in the neuropil the intial and submicron diffusion will not follow the “porous medium approach”.
It is true that we do not offer a quantitative description of how this violation of the porous medium approach would lead to an underestimation of synaptic cross-talk - we provide experimental data. However, in our appeal letter we expicitly describe this discrepancy in detail to make the reviewer aware of it, but regrettably this information never reached the reviewer.
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Reviewer #3 (Public Review):
This is an interesting paper combining several impressive techniques to argue that synaptically released glutamate is allowed to diffuse to and activate receptors at much greater distance than previously thought. iGluSnFR recordings show that glutamate released from single vesicles activates the indicator with a spatial spread (length constant) of 1.2 um, substantially farther than previous estimates based on the time course of glutamate clearance by glial transporters (PMC6725141). Similar parameters are observed with spontaneous and evoked events, large or small, or when glutamate is released via 2P uncaging. Further uncaging experiments show that both AMPARs and especially NMDARs are activated a substantial distance. AMPARs, previously thought to be recruited only within active synapses, are activated …
Reviewer #3 (Public Review):
This is an interesting paper combining several impressive techniques to argue that synaptically released glutamate is allowed to diffuse to and activate receptors at much greater distance than previously thought. iGluSnFR recordings show that glutamate released from single vesicles activates the indicator with a spatial spread (length constant) of 1.2 um, substantially farther than previous estimates based on the time course of glutamate clearance by glial transporters (PMC6725141). Similar parameters are observed with spontaneous and evoked events, large or small, or when glutamate is released via 2P uncaging. Further uncaging experiments show that both AMPARs and especially NMDARs are activated a substantial distance. AMPARs, previously thought to be recruited only within active synapses, are activated with a spatial length constant that compares quite closely with the average distance between synapses in the hippocampus. More heroic experiments and some geometric calculations show that this behavior enables neighboring synapses to interact supralinearly. The results suggest that "crosstalk" between neighboring synapses may be substantially more common than previously thought.
The experiments in this paper appear carefully performed and are analyzed thoroughly. Despite all of the quantitative rigor and careful thought, however, the authors fail to reconcile convincingly their results with what we know about neuropil structure and the laws of diffusion. There are very good data in the literature regarding the extracellular volume fraction and geometric tortuosity of the neuropil, the diffusion characteristics of glutamate and the time course of glutamate uptake. These data more or less demand that synaptically released glutamate is diluted over a much smaller spatial range than that suggested here. In the Discussion, the authors suggest that this discrepancy might reflect a simplified view of the neuropil as an isotropic diffusion medium (PMC6763864, PMC6792642, PMC6725141), whereas a more realistic network of sheets and tunnels (PMC3540825) might prolong the extracellular lifetime of neurotransmitter. I like this idea in principle, but there is no quantitative support in the paper for the claim - in fact, it seems at odds with the authors' very nice demonstration that diffusion appears to be similar in all directions (Figure 3B). I don't necessarily think a solution is within the scope of this single paper, but I would suggest that the authors acknowledge the present lack of a compelling explanation.
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Reviewer #2 (Public Review):
Matthews, Sun, McMahon et al. addresses the extent of the spread of the neurotransmitter glutamate into the extracellular space. The authors use a combination of imaging techniques, 2-photon glutamate uncaging and electrophysiology to conclude that vesicular glutamate release reaches nearby, adjacent synapses. Although this is an interesting question, and one that has been addressed many times previously, I have several technical concerns about the strength of the conclusions that reduces my enthusiasm.
-
Reviewer #1 (Public Review):
This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the interpretations …
Reviewer #1 (Public Review):
This MS combines two-photon glutamate sensing (using the iGluSnFR fluorescent probe), two-photon glutamate uncaging, two-photon calcium imaging and electrophysiology to investigate whether synaptically released glutamate activates receptors outside the synapse of release, and at neighboring synapses. The data themselves are very impressive. The authors arrive at the revolutionary conclusion that synaptically released glutamate is able to activate both NMDA and even AMPA receptors at neighboring synapses, remarkably strongly. I say revolutionary, because previous modelling has yielded diametrically opposite conclusions. The reflex would be to prefer experiment over theory, yet the modelling was based upon quite strongly constrained physical parameters that would be quite incompatible with the interpretations reported here. However, I believe the authors have failed to take into account significant technical limitations inherent in the technologies they apply. These include spatial averaging of fluorescence, possible saturation of iGluSnFR and diffusive exchange of (caged) glutamate during uncaging. As a result, the conclusion is wholly unproven. Indeed, I believe it highly probable that all of the data in favor of distal activation will prove to be consistent with synapse specificity and the presence of technical artifacts related to spatial averaging of fluorescence signals and diffusive exchange of (caged) glutamate during uncaging.
-
Evaluation Summary:
The authors address the spatial spread of glutamate outside of synapses, with the surprising conclusion that glutamate released at one synapse can strongly activate receptors at neighboring synapses. This manuscript should interest those studying neural signaling and techniques associated with that field. However, caveats of the advanced techniques used to address this difficult question limit the strength of the main conclusion.
(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. The reviewers remained anonymous to the authors.)
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