GABABR silencing of nerve terminals
Curation statements for this article:-
Curated by eLife
eLife assessment
The authors revisit fundamentals of synaptic transmission using a combination of advanced optical methods capable of visualizing calcium influx and neurotransmitter release at single release sites. By doing so, the authors present evidence for silencing of neurotransmitter release at single release sites as a function of external calcium. The data have relevance to a wide range of phenomena including neural plasticity and inhibitory modulation of synaptic communication.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca 2+ ) influx in the active zone. Here, using quantitative analysis of both single bouton Ca 2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca 2+ influx and the concentration of external Ca 2+ ([Ca 2+ ] e ). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca 2+ ] e , 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or-none fashion at the single synapse level when operating at the physiological set point.
Article activity feed
-
-
Author Response
Reviewer #2 (Public Review):
Throughout the manuscript, the authors aim to distinguish signal from the lack of it. All conclusions depend on the success of this process. In such an endeavor, the sensitivity of the applied methods is critical. Thus, the authors must use the most sensitive tools to draw meaningful conclusions. The latest iGluSnFR has amazing sensitivity allowing the detection of single AP-evoked responses. This is not the case for vGpH, which requires hundred APs to get a meaningful signal. Similar, synthetic Ca2+ dyes have much better dynamic range, linearity and sensitivity compared to GCaMP6f.
The rate of silent boutons at 2 mM [Ca2+]e is lower for a single AP compared to 20 or 200 APs. The overall failure rate cannot be increased with increasing the number of APs. This clearly indicates a …
Author Response
Reviewer #2 (Public Review):
Throughout the manuscript, the authors aim to distinguish signal from the lack of it. All conclusions depend on the success of this process. In such an endeavor, the sensitivity of the applied methods is critical. Thus, the authors must use the most sensitive tools to draw meaningful conclusions. The latest iGluSnFR has amazing sensitivity allowing the detection of single AP-evoked responses. This is not the case for vGpH, which requires hundred APs to get a meaningful signal. Similar, synthetic Ca2+ dyes have much better dynamic range, linearity and sensitivity compared to GCaMP6f.
The rate of silent boutons at 2 mM [Ca2+]e is lower for a single AP compared to 20 or 200 APs. The overall failure rate cannot be increased with increasing the number of APs. This clearly indicates a technical issue (e.g. insufficient sensitivity of vGpH and GCaMP6f).
We thank the reviewer for raising this concern. We attribute the relatively lower rate of silencing with 1 AP in [Ca2+]e 2.0 mM in neurons expressing iGluSnFr to its sensitivity to detect glutamate exocytosed from neighboring, possibly non-transfected terminals. This limitation is described in the manuscript (page 7, line 26 – page 8, line 5). The overall agreement in the proportion of silencing with iGluSnFr compared to physin-GCaMP or vGpH at lower [Ca2+]e, where the contributions from neighboring terminals is likely greatly diminished, supports this interpretation.
The authors used three different measuring tools and used three different stimulation protocols, making the interpretation of the data challenging. It is impossible to tell how the failure rate changes from 1 to 20 APs without knowing the release probability, the pool size, depletion, recovery of SVs, and facilitation. These are all unknown.
In an ideal world, a measure of release probability during a train of stimuli at varied [Ca2+]e would provide the most insight, but this is difficult to achieve with any of the existing methods, including the remarkable new iGluSnFR. The challenge we face is, for our approach, it is impossible to exclude signals from neighboring axons that are closely packed near the axon harboring the indicator. This limitation is described in the manuscript (page 7, line 26 – page 8, line 5). Given this, we felt that showing that silencing can be revealed with all the different techniques was the most conservative approach to address the issue. Because we have focused on this phenomenon, the number of APs is experimentally important only to ensure an adequate response could be detected. We have also included, in the discussion, an acknowledgement of the possibility that we are failing to detect minimal Ca2+ entry (see response to #8 from the synthesized review).
The last experiment with the GABAB agonist has little novelty in its present form. The authors demonstrate that GABAB agonism increases the rate of silent terminals. The interesting issue would be to reveal how the effect of GABAB activation depends on the [Ca2+]e. This information is essential to see whether there is indeed a shoulder in its effectiveness curve.
We are grateful to the reviewer for this recommendation and we have performed additional experiments (see response to #7 from the synthesized review).
The authors refer to a theoretical set-point in [Ca2+]e below which the function of the terminals is fundamentally different. From the presented experiments, the reviewer does not see any data that is inconsistent with a continuum. 'Thus, as with Ca2+ influx, SV recycling is modulated in an all-or-none manner by modest changes in [Ca2+]e around the physiological set point.' This statement is not supported by the data. The reviewer cannot see a set point.
We appreciate the reviewer’s criticism and wish to clarify that we mean the normal physiologic [Ca2+]e in the CSF. We have changed the text to clarify this point (page 7, line 20).
-
eLife assessment
The authors revisit fundamentals of synaptic transmission using a combination of advanced optical methods capable of visualizing calcium influx and neurotransmitter release at single release sites. By doing so, the authors present evidence for silencing of neurotransmitter release at single release sites as a function of external calcium. The data have relevance to a wide range of phenomena including neural plasticity and inhibitory modulation of synaptic communication.
-
Reviewer #1 (Public Review):
This is a fascinating effort from the Ryan laboratory, revisiting fundamental issues of calcium-dependent release probability at cultured synapses. The authors point out that our basic understanding of mammalian synapses rests on a foundation of older research that was not acquired at physiological temperature, and represented a statistical interpretation of data acquired electrophysiologically without direct knowledge of release at individual active zones. The authors employ techniques of calcium imaging and glutamate sensing and argue that single synapses can be 'silenced' by a moderate drop in extracellular calcium, a drop that is within the range of calcium channel inhibition following activation of GABAergic signaling. While fascinating, the conclusions are most powerful when the data can be distilled …
Reviewer #1 (Public Review):
This is a fascinating effort from the Ryan laboratory, revisiting fundamental issues of calcium-dependent release probability at cultured synapses. The authors point out that our basic understanding of mammalian synapses rests on a foundation of older research that was not acquired at physiological temperature, and represented a statistical interpretation of data acquired electrophysiologically without direct knowledge of release at individual active zones. The authors employ techniques of calcium imaging and glutamate sensing and argue that single synapses can be 'silenced' by a moderate drop in extracellular calcium, a drop that is within the range of calcium channel inhibition following activation of GABAergic signaling. While fascinating, the conclusions are most powerful when the data can be distilled to direct observation of single release sites and this is not uniformly the case.
-
Reviewer #2 (Public Review):
Throughout the manuscript, the authors aim to distinguish signal from the lack of it. All conclusions depend on the success of this process. In such an endeavor, the sensitivity of the applied methods is critical. Thus, the authors must use the most sensitive tools to draw meaningful conclusions. The latest iGluSnFR has amazing sensitivity allowing the detection of single AP-evoked responses. This is not the case for vGpH, which requires hundred APs to get a meaningful signal. Similar, synthetic Ca2+ dyes have much better dynamic range, linearity and sensitivity compared to GCaMP6f.
The rate of silent boutons at 2 mM [Ca2+]e is lower for a single AP compared to 20 or 200 APs. The overall failure rate cannot be increased with increasing the number of APs. This clearly indicates a technical issue (e.g. …
Reviewer #2 (Public Review):
Throughout the manuscript, the authors aim to distinguish signal from the lack of it. All conclusions depend on the success of this process. In such an endeavor, the sensitivity of the applied methods is critical. Thus, the authors must use the most sensitive tools to draw meaningful conclusions. The latest iGluSnFR has amazing sensitivity allowing the detection of single AP-evoked responses. This is not the case for vGpH, which requires hundred APs to get a meaningful signal. Similar, synthetic Ca2+ dyes have much better dynamic range, linearity and sensitivity compared to GCaMP6f.
The rate of silent boutons at 2 mM [Ca2+]e is lower for a single AP compared to 20 or 200 APs. The overall failure rate cannot be increased with increasing the number of APs. This clearly indicates a technical issue (e.g. insufficient sensitivity of vGpH and GCaMP6f).
The authors used three different measuring tools and used three different stimulation protocols, making the interpretation of the data challenging. It is impossible to tell how the failure rate changes from 1 to 20 APs without knowing the release probability, the pool size, depletion, recovery of SVs, and facilitation. These are all unknown.
The last experiment with the GABAB agonist has little novelty in its present form. The authors demonstrate that GABAB agonism increases the rate of silent terminals. The interesting issue would be to reveal how the effect of GABAB activation depends on the [Ca2+]e. This information is essential to see whether there is indeed a shoulder in its effectiveness curve.
The authors refer to a theoretical set-point in [Ca2+]e below which the function of the terminals is fundamentally different. From the presented experiments, the reviewer does not see any data that is inconsistent with a continuum. 'Thus, as with Ca2+ influx, SV recycling is modulated in an all-or-none manner by modest changes in [Ca2+]e around the physiological set point.' This statement is not supported by the data. The reviewer cannot see a set point.
-
Reviewer #3 (Public Review):
In this study Cook and Ryan examine, at physiological temperatures, the sensitivity of neurotransmitter release to external calcium concentrations close to physiological ones. Using hippocampal neurons in culture, field potential-based stimulation, a spatially confined genetically encoded calcium indicator (GCaMP6f) as well as fluorescent reporters of exocytosis and extracellular glutamate, the authors show that as extracellular calcium concentrations are reduced from 2.0, to 1.2 and finally to 0.8 mM, a disproportional fraction of presynaptic terminals cease to respond, as evidenced by no elevations in intracellular calcium concentrations, no detectable exocytosis or changes in extracellular glutamate. The phenomenon is quantitively modulated by blocking particular types of calcium channels, but is …
Reviewer #3 (Public Review):
In this study Cook and Ryan examine, at physiological temperatures, the sensitivity of neurotransmitter release to external calcium concentrations close to physiological ones. Using hippocampal neurons in culture, field potential-based stimulation, a spatially confined genetically encoded calcium indicator (GCaMP6f) as well as fluorescent reporters of exocytosis and extracellular glutamate, the authors show that as extracellular calcium concentrations are reduced from 2.0, to 1.2 and finally to 0.8 mM, a disproportional fraction of presynaptic terminals cease to respond, as evidenced by no elevations in intracellular calcium concentrations, no detectable exocytosis or changes in extracellular glutamate. The phenomenon is quantitively modulated by blocking particular types of calcium channels, but is qualitatively conserved across all tested conditions. Finally, the authors show that effects of lower extracellular calcium concentrations can be mimicked by applying Baclofen, an agonist of type B GABA receptors. The authors reveal the sensitivity of all-or none calcium influx and exocytosis near extracellular calcium physiological set points and highlight the potential importance of this sensitivity as an effective control point for neural circuit modulation.
The findings described in the manuscript are potentially important as they seem to uncover a new, yet undescribed, all-or none (binary) phenomenon in the field of synaptic neuroscience, that is, of individual presynaptic terminals moving between two 'states' - 'active' and 'silenced'- which are set somehow by levels of extracellular calcium concentrations. Moreover, this dependency is observed at extracellular calcium concentrations that are quite close to the physiological concentration set point. The use of multiple reporters (intracellular calcium concentrations, synaptic vesicle fusion and extracellular glutamate) strengthens the validity of the observations.
On the other hand, there are two major points that need to be addressed.
The first is that alternative explanations should be ruled out more convincingly, first and foremost the matter of membrane excitability. Two observations are relevant here: The qualitative preservation of the phenomenon when two types of voltage gated calcium channels are blocked separately, and the large heterogeneity of the % of silenced boutons among neurons at a given extracellular calcium concentrations, which is at least as great as the range of modulation of the % of silenced synapses by extracellular calcium concentrations at single neurons. One then wonders if the findings might be attributed to a) the fidelity of the field potential-based stimulation system, that is, the degree to which neurons track the stimuli trains; b) the heterogeneity of neurons in this regard, c) this fidelity at different extracellular calcium concentrations for different neurons, and d) the identity of presynaptic sites analyzed in one run (are they all part of the same axon?). Along these lines, there is an assumption that the field potential-based stimulation system is the sole driver of excitation in these networks, which is reasonable given that excitatory synaptic transmission is mostly blocked pharmacologically (by CNQX and APV). Inhibitory transmission, however, was not blocked and thus, there is no guarantee that the inhibitory input neurons receive and its modulation by extracellular calcium does affect the degree to which neurons fire precisely and reliably at 20 Hz at all conditions. If it could be shown, at least for a substantial subset of the data, that all terminals analyzed for a particular neuron are part of an unambiguously identified axon stretch, with no branches (potential conduction failure points) and still demonstrate the claimed heterogeneity, this potential confound would be less of an issue.
The second issue relates to the ties made to neuromodulation. In spite of the title, introduction and discussion, not a single neuromodulator (such as dopamine, acetylcholine, noradrenaline, serotonin) was tested, only baclofen, which as a derivative of GABA, activates GABAB receptors, not receptors of canonical neuromodulators. The title of this manuscript is therefore not appropriate.
-
