Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Keine et al study the role of the RhoGTPase Rac1 in neurotransmitter release by ablating this protein at an age when synapses are in an almost mature stage. They describe an increase in synaptic strength, which they interpret as an increase in release probability or fusogenicity of synaptic vesicles. They also describe subtle effects in the timing of release, which point towards a mild defect in positional priming. The study delivers important information on the role of Rac1.
(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 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1’s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.
Article activity feed
-
-
-
Author Response
Reviewer #1 (Public Review):
This study uses the mouse calyx of Held synapse as a model to explore the presynaptic role of rac1, a regulator of actin signaling in the brain. Many of the now-classical methods and theory pioneered by Neher and colleagues are brought to bear on this problem. Additionally, the authors were able to make a cell-specific knockout of rac1 by developing a novel viral construct to express cre in the globular bushy cells of the cochlear nucleus; by doing this in a rac1 floxed mouse, they were able to KO rac1 in these neurons starting at around P14. The authors found that KO of rac1 enhanced EPSC amplitude, vesicle release probability, quantal release rates, EPSC onset time and jitter during high-frequency activity, and fast recovery rates from depression. Because the calyx synapses are the …
Author Response
Reviewer #1 (Public Review):
This study uses the mouse calyx of Held synapse as a model to explore the presynaptic role of rac1, a regulator of actin signaling in the brain. Many of the now-classical methods and theory pioneered by Neher and colleagues are brought to bear on this problem. Additionally, the authors were able to make a cell-specific knockout of rac1 by developing a novel viral construct to express cre in the globular bushy cells of the cochlear nucleus; by doing this in a rac1 floxed mouse, they were able to KO rac1 in these neurons starting at around P14. The authors found that KO of rac1 enhanced EPSC amplitude, vesicle release probability, quantal release rates, EPSC onset time and jitter during high-frequency activity, and fast recovery rates from depression. Because the calyx synapses are the largest and most reliable of central nerve terminals, all these various effects had no effect on suprathreshold transmission during 'in vivo-like' stimulus protocols. Moreover, there was no effect morphologically on the synapse. Through some unavoidably serpentine reasoning, the authors suggest that loss of rac1 affects the so-called molecular priming of vesicles, possibly due to a restructuring of actin barriers at the active zone. The experimental analysis is at a very high level, and the work is definitely an important contribution to the field of presynaptic physiology and biophysics. It will be important to test the effects of the KO on other synapses that are not such high-performers as the calyx, and this direction might reveal significant effects on information processing by altered rac1 expression.
We thank the reviewer for their comments and view that our work is an important contribution to the field of presynaptic physiology and biophysics.
Major points:
- The measurement of onset delay was used to test whether rac1-/- affects positional priming. While there is a clear effect of the KO on the latency to EPSC onset, there is no singular interpretation one can take, due to the ambiguity of the 'onset delay'. Note that in the Results authors state Lines 201-203: "The time between presynaptic AP and EPSC onset (EPSC onset delay) is determined by the distance between SVs and VGCC which defines the time it takes for Ca2+ to bind to the Ca2+ sensor and trigger SV release (Fedchyshyn and Wang, 2007)." However, in Methods "The duration between stimulus and EPSC onset was defined as EPSC onset delay." Thus the 'onset' measured is not between presynaptic spike and EPSC but from axonal stimulus and EPSC. KO of rac might also affect spike generation, spike conduction, calcium channel function, etc. Indeed some additional options are offered in the Discussion. Since the change in onset is ~100usec at most, a number of small factors all could contribute here. Moreover, the authors conclude that the KO does NOT affect positional priming since they would have expected the onset to shorten, given the other enhancements observed in earlier sections.
It seems to me that all the authors can really conclude is that the onset shifted and they do not know why. If onset is driven by multiple factors, and differentially affected in the KO, then all bets are off. Thus, data in this section might be removed, or at least the authors could further qualify their interpretations given this ambiguity.
We have further qualified and clarified our interpretations of the EPSC onset measurement. To do so, we have added additional text to the Discussion (see lines 475-491). We would like to emphasize that we do not see a statistically significant change in EPSC1 onset delay and EPSC onset delays during 50 Hz train stimuli between the Rac1+/+- and Rac1−/− synapses but rather an activity-dependent increase in EPSC onset delays in Rac1−/− synapses during 500 Hz stimulation. It is important to note that based on these data, it is less likely that changes in spike generation, spike conduction, or calcium channel function are responsible for the change in EPSC onset delay. If SVs were closer to CaV2.1 channels, we would expect shorter initial EPSC onset delay time or shorter EPSC onset delay times during 50 Hz stimulation. However, changes in spike generation, spike conduction or calcium channel function could contribute to the increase in the EPSC onset delay at 500 Hz. Finally, it is important to note that EPSC onset delay increase during 50 Hz and 500 Hz stimulation in Rac1+/+ synapses indicating an activity-dependent regulation. However, this activity-dependent increase was pronounced in Rac1−/− synapses during both 50 Hz and 500 Hz stimulation (Fig 4B1-B3).
- If the idea is that the loss of Rac1 leads to a reduced actin barrier at the active zone, is there an ultrastructural way to visualize this, labeling for actin for example? Authors conclude that new techniques are needed, but perhaps this is 'just' an EM question.
We are not aware of a method for ultrastructural visualization of actin and SV distributions relative to the plasma membrane. To do so requires specific labeling and detection of actin filaments while visualizing SVs using EM. While EM on samples prepared by high-pressure freeze with freeze substitution allows for detection of filamentous structures near the AZ, the molecular identity of these filamentous structures would remain uncertain. Super-resolution microscopy is amenable to immunohistochemical techniques to label actin, but visualizing SVs in 3D using super-resolution is a major technical challenge. Furthermore, changes in SV docking on the scale of 1-2 nanometers are correlated with severe changes in SV release, therefore we would need to be able to quantify structural changes at this level of resolution. Currently, we are not aware of any study or report that has analyzed SV docking or reported changes on the scale of 1-2 nm using super-resolution light microscopy. It might be possible to use expansion microscopy to achieve such resolution but the respective protocols would need to be established for the calyx synapse. In addition, it is proposed that the regulation of actin filaments is transient and happens on very fast time scales which complicates their investigation by conventional methods (O'Neil et al., 2021). Thus, even if we were able to solve all these technical hurdles, it is well possible to miss potential differences even if we were able to label actin. Therefore, while we agree that having this type of ultrastructural data available would strongly strengthen our hypothesis, the development of the techniques and protocols needed to perform these types of experiments would likely require many months if not years.
- Authors use 1 mM kynurenic acid in the bath to avoid postsynaptic receptor saturation. But since this is a competitive antagonist and since the KO shows a large increase in release, could saturation or desensitization have been enhanced in the KO? This would affect the interpretation of recovery rates in the KO, which are quite fast.
We agree with the reviewer that differences in saturation or desensitization could potentially impact the measured recovery time course in Rac1−/−. However, we think this is unlikely because of the following reasons: Desensitization and saturation of synaptic AMPARs is strongly reduced during calyx synapse maturation (Taschenberger et al., 2002; Taschenberger et al., 2005). We recorded from >P28 calyx synapses which exhibit a claw-like, fenestrated terminal morphology offering many diffusional exits for released glutamate which is expected to speed up transmitter clearance and therefore reduce postsynaptic effects (Taschenberger et al., 2005; Yang et al., 2021). We used 1 mM Kynurenic acid in the external bath solution which resulted in a ~90% reduction in EPSC amplitude in both Rac1+/+ and Rac1−/−, which is comparable to previous reports (e.g. Lipstein et al., 2021). In our study, we performed all experiments in 1.2 mM Ca2+ and at body temperature which further reduces EPSC amplitudes and minimizes potential receptor saturation and desensitization compared to 2 mM Ca2+ at room temperature. Time constants of recovery from desensitization at the calyx are between 30 ms at P14-P16 (Joshi et al., 2004) and 16 ms at P21 (Koike-Tani et al., 2008), both measured at room temperature. It is conceivable that the recovery from desensitization at P30 and at physiological temperature will be significantly shorter. Since we observed the largest effect in recovery between 1 and 4 seconds, this is at least two orders of magnitude slower than the recovery from desensitization could likely account for. Finally, our numerical simulations are consistent with the possibility of faster recovery rates observed in Rac1−/− being a direct consequence of changes in SV priming. This faster pool replenishment likely also enabled increased steady-state EPSC amplitudes at 50 Hz in Rac1−/− synapses. The fact that we were able to measure enhanced steady-state release in Rac1−/− argues against steady-state EPSC amplitudes being limited by AMPARs desensitization.
Reviewer #2 (Public Review):
The aim of the study is an improved understanding of the role of the RhoGTPase Rac1 in neurotransmitter release beyond the known roles in synaptogenesis and postsynaptic function. To this end, Rac1 is ablated at P12 (when synapse development has largely progressed to maturation) and transmission is studied at the adult stage (P28 onwards). The study reports a number of interesting findings, in particular, a large increase in synaptic strength, which is interpreted as an '... increased release probability, which results in faster SV replenishment'. It is not clear whether this statement is supposed to suggest a causal relationship or just a correlation between the two parameters. By and large, the discussion of results is somewhat fuzzy with respect to the distinction between release itself (as characterized by release probability) and priming steps, which precede release.
Besides, the authors present valuable data on Rac1-dependent timing and synchronicity of neurotransmitter release, which point towards a role of Rac1 in 'positional priming', i. e. the proper localization of synaptic vesicles relative to Ca-channels.
We thank the reviewer for pointing out that our study present valuable data on Rac1-dependent timing and synchronicity of neurotransmitter release.
-
-
Evaluation Summary:
Keine et al study the role of the RhoGTPase Rac1 in neurotransmitter release by ablating this protein at an age when synapses are in an almost mature stage. They describe an increase in synaptic strength, which they interpret as an increase in release probability or fusogenicity of synaptic vesicles. They also describe subtle effects in the timing of release, which point towards a mild defect in positional priming. The study delivers important information on the role of Rac1.
(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 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This study uses the mouse calyx of Held synapse as a model to explore the presynaptic role of rac1, a regulator of actin signaling in the brain. Many of the now-classical methods and theory pioneered by Neher and colleagues are brought to bear on this problem. Additionally, the authors were able to make a cell-specific knockout of rac1 by developing a novel viral construct to express cre in the globular bushy cells of the cochlear nucleus; by doing this in a rac1 floxed mouse, they were able to KO rac1 in these neurons starting at around P14. The authors found that KO of rac1 enhanced EPSC amplitude, vesicle release probability, quantal release rates, EPSC onset time and jitter during high-frequency activity, and fast recovery rates from depression. Because the calyx synapses are the largest and most …
Reviewer #1 (Public Review):
This study uses the mouse calyx of Held synapse as a model to explore the presynaptic role of rac1, a regulator of actin signaling in the brain. Many of the now-classical methods and theory pioneered by Neher and colleagues are brought to bear on this problem. Additionally, the authors were able to make a cell-specific knockout of rac1 by developing a novel viral construct to express cre in the globular bushy cells of the cochlear nucleus; by doing this in a rac1 floxed mouse, they were able to KO rac1 in these neurons starting at around P14. The authors found that KO of rac1 enhanced EPSC amplitude, vesicle release probability, quantal release rates, EPSC onset time and jitter during high-frequency activity, and fast recovery rates from depression. Because the calyx synapses are the largest and most reliable of central nerve terminals, all these various effects had no effect on suprathreshold transmission during 'in vivo-like' stimulus protocols. Moreover, there was no effect morphologically on the synapse. Through some unavoidably serpentine reasoning, the authors suggest that loss of rac1 affects the so-called molecular priming of vesicles, possibly due to a restructuring of actin barriers at the active zone. The experimental analysis is at a very high level, and the work is definitely an important contribution to the field of presynaptic physiology and biophysics. It will be important to test the effects of the KO on other synapses that are not such high-performers as the calyx, and this direction might reveal significant effects on information processing by altered rac1 expression.
Major points:
1. The measurement of onset delay was used to test whether rac1-/- affects positional priming. While there is a clear effect of the KO on the latency to EPSC onset, there is no singular interpretation one can take, due to the ambiguity of the 'onset delay'. Note that in the Results authors state Lines 201-203: "The time between presynaptic AP and EPSC onset (EPSC onset delay) is determined by the distance between SVs and VGCC which defines the time it takes for Ca2+ to bind to the Ca2+ sensor and trigger SV release (Fedchyshyn and Wang, 2007)." However, in Methods "The duration between stimulus and EPSC onset was defined as EPSC onset delay." Thus the 'onset' measured is not between presynaptic spike and EPSC but from axonal stimulus and EPSC. KO of rac might also affect spike generation, spike conduction, calcium channel function, etc. Indeed some additional options are offered in the Discussion. Since the change in onset is ~100usec at most, a number of small factors all could contribute here. Moreover, the authors conclude that the KO does NOT affect positional priming since they would have expected the onset to shorten, given the other enhancements observed in earlier sections.
It seems to me that all the authors can really conclude is that the onset shifted and they do not know why. If onset is driven by multiple factors, and differentially affected in the KO, then all bets are off. Thus, data in this section might be removed, or at least the authors could further qualify their interpretations given this ambiguity.2. If the idea is that the loss of Rac1 leads to a reduced actin barrier at the active zone, is there an ultrastructural way to visualize this, labeling for actin for example? Authors conclude that new techniques are needed, but perhaps this is 'just' an EM question.
3. Authors use 1 mM kynurenic acid in the bath to avoid postsynaptic receptor saturation. But since this is a competitive antagonist and since the KO shows a large increase in release, could saturation or desensitization have been enhanced in the KO? This would affect the interpretation of recovery rates in the KO, which are quite fast.
-
Reviewer #2 (Public Review):
The aim of the study is an improved understanding of the role of the RhoGTPase Rac1 in neurotransmitter release beyond the known roles in synaptogenesis and postsynaptic function. To this end, Rac1 is ablated at P12 (when synapse development has largely progressed to maturation) and transmission is studied at the adult stage (P28 onwards). The study reports a number of interesting findings, in particular, a large increase in synaptic strength, which is interpreted as an '... increased release probability, which results in faster SV replenishment'. It is not clear whether this statement is supposed to suggest a causal relationship or just a correlation between the two parameters. By and large, the discussion of results is somewhat fuzzy with respect to the distinction between release itself (as characterized …
Reviewer #2 (Public Review):
The aim of the study is an improved understanding of the role of the RhoGTPase Rac1 in neurotransmitter release beyond the known roles in synaptogenesis and postsynaptic function. To this end, Rac1 is ablated at P12 (when synapse development has largely progressed to maturation) and transmission is studied at the adult stage (P28 onwards). The study reports a number of interesting findings, in particular, a large increase in synaptic strength, which is interpreted as an '... increased release probability, which results in faster SV replenishment'. It is not clear whether this statement is supposed to suggest a causal relationship or just a correlation between the two parameters. By and large, the discussion of results is somewhat fuzzy with respect to the distinction between release itself (as characterized by release probability) and priming steps, which precede release.
Besides, the authors present valuable data on Rac1-dependent timing and synchronicity of neurotransmitter release, which point towards a role of Rac1 in 'positional priming', i. e. the proper localization of synaptic vesicles relative to Ca-channels.
-
-