Analogue signaling of somatodendritic synaptic activity to axon enhances GABA release in young cerebellar molecular layer interneurons
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Small subthreshold dendritic-somatic depolarizations can propagate to presynaptic nerve endings and may modulate transmitter release, but the mechanisms of this modulation remain poorly understood because the technical challenge of recording from small bouton synapse. Here the authors directly record from small cerebellar bouton terminals In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs recorded in the Purkinje cells and change synaptic plasticity during AP trains. In order to address mechanisms of such presynaptic modulation, the authors measure presynaptic AP waveforms via cell attached recordings and found these very stable. On the other hand, presynaptic ICa(V) directly recorded in voltage-clamped MLI boutons facilitated in response to small pre-depolarizations and such facilitated ICa(V) produced larger IPSCs in paired recordings of MLI boutons and coupled Purkinje cells. The authors propose that an accumulation of partially gated channels during small presynaptic depolarizations is able to produce more rapid gating of VGCCs during the AP waveform on arrival of an invading presynaptic AP.
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Abstract
Axons are equipped with the digital signaling capacity by which they generate and faithfully propagate action potentials (APs), and also with the analogue signaling capacity by which subthreshold activity in dendrites and soma is transmitted down the axon. Despite intense work, the extent and physiological role for subthreshold synaptic activity reaching the presynaptic boutons has remained elusive because of the technical limitation to record from them. To address this issue, we made simultaneous patch-clamp recordings from the presynaptic varicosities of cerebellar GABAergic interneurons together with their parent soma or postsynaptic target cells in young rat slices and/or primary cultures. Our tour-de-force direct functional dissection indicates that the somatodendritic spontaneous excitatory synaptic potentials are transmitted down the axon for significant distances, depolarizing presynaptic boutons. These analogously transmitted excitatory synaptic potentials augment presynaptic Ca ++ influx upon arrival of an immediately following AP through a mechanism that involves a voltage-dependent priming of the Ca ++ channels, leading to an increase in GABA release, without any modification in the presynaptic AP waveform or residual Ca ++ . Our work highlights the role of the axon in synaptic integration.
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eLife assessment
Small subthreshold dendritic-somatic depolarizations can propagate to presynaptic nerve endings and may modulate transmitter release, but the mechanisms of this modulation remain poorly understood because the technical challenge of recording from small bouton synapse. Here the authors directly record from small cerebellar bouton terminals In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs …
eLife assessment
Small subthreshold dendritic-somatic depolarizations can propagate to presynaptic nerve endings and may modulate transmitter release, but the mechanisms of this modulation remain poorly understood because the technical challenge of recording from small bouton synapse. Here the authors directly record from small cerebellar bouton terminals In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs recorded in the Purkinje cells and change synaptic plasticity during AP trains. In order to address mechanisms of such presynaptic modulation, the authors measure presynaptic AP waveforms via cell attached recordings and found these very stable. On the other hand, presynaptic ICa(V) directly recorded in voltage-clamped MLI boutons facilitated in response to small pre-depolarizations and such facilitated ICa(V) produced larger IPSCs in paired recordings of MLI boutons and coupled Purkinje cells. The authors propose that an accumulation of partially gated channels during small presynaptic depolarizations is able to produce more rapid gating of VGCCs during the AP waveform on arrival of an invading presynaptic AP.
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Reviewer #1 (Public Review):
This study represents in exciting collaboration between two young independent scientists in Uruguay and Japan. Trigo and Kawaguchi provide evidence for the presynaptic modulation of the opening-probability of calcium channels as a major mechanism of digital-analog coupling in immature cerebellar molecular layer interneurons (MLI). Applying a combination of electrophysiological methods including direct axonal whole-cell patch-clamp recordings and glutamate photolysis in acute brain slices and dissociated cultured neurons, the authors provide the following empirical findings: 1) Spontaneous and evoked EPSPs are reliably transmitted into the presynaptic compartment. The amplitude of the spontaneous EPSPs decayed with a length constant of 180 µm in the axon. 2) Physiologically relevant short and subthreshold (< …
Reviewer #1 (Public Review):
This study represents in exciting collaboration between two young independent scientists in Uruguay and Japan. Trigo and Kawaguchi provide evidence for the presynaptic modulation of the opening-probability of calcium channels as a major mechanism of digital-analog coupling in immature cerebellar molecular layer interneurons (MLI). Applying a combination of electrophysiological methods including direct axonal whole-cell patch-clamp recordings and glutamate photolysis in acute brain slices and dissociated cultured neurons, the authors provide the following empirical findings: 1) Spontaneous and evoked EPSPs are reliably transmitted into the presynaptic compartment. The amplitude of the spontaneous EPSPs decayed with a length constant of 180 µm in the axon. 2) Physiologically relevant short and subthreshold (< 10 mV) depolarizations before action potentials ('pre-AP') increase the release probability and subsequently short-term depression at the MLI-Purkinje cell synapse without changing the duration of APs and just a minor reduction in amplitude of APs (< 10%). 3) The pre-AP subthreshold depolarizations subsequently increase the amplitude of AP-induced presynaptic calcium currents and GABAergic postsynaptic currents. 4) A short interval of only 3 ms duration between the pre-AP depolarization and the AP blocks the analog coupling. 5) A biophysical model of presynaptic calcium channel gating is proposed, which involves depolarization-induced intermediate gating steps that increase the probability of activating the channels during the AP.
A particular strength of this study is the large data set of technically very challenging direct recordings from small presynaptic terminals. The proposed mechanism provides an innovative explanation for the experimental findings. The most innovative experiments might be those with a 3-ms-gap between the pre-APs and APs. At this synapse, elevated residual intracellular calcium concentration was previously shown to mediate analog coding (https://doi.org/10.1523/JNEUROSCI.5127-10.2011). However, the elevated residual calcium cannot explain the surprising block of analog coding by a 3-ms-gap in the depolarization, because intracellular calcium signals decay with kinetics in the range of 100 ms. Both mechanisms (residual calcium and priming of calcium channels) are probably operating in parallel and future studies should resolve the exact interplay of both mechanisms. A potential weakness of the study is that the proposed priming of calcium channels is not shown explicitly to be able to explain the experimental data. Quantitive simulations of calcium channel gating states were only performed in steady-state but not in a time-dependent manner during pre-APs and APs.
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Reviewer #2 (Public Review):
This study used direct recording from the soma, the terminal and the postsynaptic cell in cerebellar inter-neuron- Purkinje cell synapses. The authors nicely showed that action potentials travel reliably from the soma to the axon. In addition, they showed that the postsynaptic responses elicited at the dendrites reliably traveled along the axon. Such sub-threshold potential could potentiate transmitter release in short-term (for tens of ms at most), by "priming" Ca channels and accelerating activation kinetics of Ca channels. Results are based on the technically demanding electrophysiological technique and are in general. The study directly solves the mechanism of short-term facilitation induced by sub-threshold depolarization.
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Reviewer #3 (Public Review):
Trigo & Kawaguchi study how small somatic subthreshold depolarizations that do not trigger full blown APs can propagate to presynaptic endings and modulate transmitter release. To this end they directly recorded from small cerebellar MLI boutons. In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs recorded in the Purkinje cells and change synaptic plasticity during AP trains. In order …
Reviewer #3 (Public Review):
Trigo & Kawaguchi study how small somatic subthreshold depolarizations that do not trigger full blown APs can propagate to presynaptic endings and modulate transmitter release. To this end they directly recorded from small cerebellar MLI boutons. In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs recorded in the Purkinje cells and change synaptic plasticity during AP trains. In order to address mechanisms of such presynaptic modulation, the authors measure presynaptic AP waveforms via cell attached recordings and found these very stable. On the other hand, presynaptic ICa(V) directly recorded in voltage-clamped MLI boutons facilitated in response to small pre-depolarizations and such facilitated ICa(V) produced larger IPSCs in paired recordings of MLI boutons and coupled Purkinje cells. The authors propose that an accumulation of partially gated channels during small presynaptic depolarizations is able to produce more rapid gating of VGCCs during the AP waveform on arrival of an invading presynaptic AP.
Electrotonic coupling between soma and presynaptic endings to the extent that small subthreshold depolarizations such as synaptic potentials can travel to the bouton has been demonstrated before. However direct quantification of such coupling is difficult because of the small size of presynaptic compartments. Trigo & Kawaguchi have now pioneered such very challenging direct presynaptic recordings in the form of recordings of MLI soma and bouton pairs or paired pre- and postsynaptic recordings.
The data is convincing and I do not see a need for additional experiments. But the manuscript in its present form falls short with respect to the presentation and discussion of the data. The authors conclusion about the mechanism of presynaptic ICa(V) facilitation should be verified with proper kinetic simulations using a kinetic scheme such as that proposed by Li, Bischofberger & Jonas (2007) J.Neurosci. which should be adapted to the presynaptic ICa(V) in MLI boutons. This would strengthen the manuscript which otherwise, regarding mechanisms, remains somewhat speculative.
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