Kv3.3 subunits control presynaptic action potential waveform and neurotransmitter release at a central excitatory synapse
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Evaluation Summary:
The authors make a compelling case for a special type of potassium channel (Kv3.3) in the control of the presynaptic AP spike waveforms. Importantly, mice that lack Kv3.3 showed auditory response deficits, including increases in pre-synaptic AP halfwidth, AP latency, AP jitter and spontaneous activity. The in vivo recordings are impressive. The study contains an extensive data set and makes a compelling argument for the uniquely important role for Kv3.3 in synaptic transmission. Overall, the findings will significantly advance our understanding of the calyx of Held and the neural circuit of sound processing, as well as the functions of Kv3 channels.
(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 #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Kv3 potassium currents mediate rapid repolarisation of action potentials (APs), supporting fast spikes and high repetition rates. Of the four Kv3 gene family members, Kv3.1 and Kv3.3 are highly expressed in the auditory brainstem and we exploited this to test for subunit-specific roles at the calyx of Held presynaptic terminal in the mouse. Deletion of Kv3.3 (but not Kv3.1) reduced presynaptic Kv3 channel immunolabelling, increased presynaptic AP duration and facilitated excitatory transmitter release; which in turn enhanced short-term depression during high-frequency transmission. The response to sound was delayed in the Kv3.3KO, with higher spontaneous and lower evoked firing, thereby reducing signal-to-noise ratio. Computational modelling showed that the enhanced EPSC and short-term depression in the Kv3.3KO reflected increased vesicle release probability and accelerated activity-dependent vesicle replenishment. We conclude that Kv3.3 mediates fast repolarisation for short precise APs, conserving transmission during sustained high-frequency activity at this glutamatergic excitatory synapse.
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Evaluation Summary:
The authors make a compelling case for a special type of potassium channel (Kv3.3) in the control of the presynaptic AP spike waveforms. Importantly, mice that lack Kv3.3 showed auditory response deficits, including increases in pre-synaptic AP halfwidth, AP latency, AP jitter and spontaneous activity. The in vivo recordings are impressive. The study contains an extensive data set and makes a compelling argument for the uniquely important role for Kv3.3 in synaptic transmission. Overall, the findings will significantly advance our understanding of the calyx of Held and the neural circuit of sound processing, as well as the functions of Kv3 channels.
(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 …
Evaluation Summary:
The authors make a compelling case for a special type of potassium channel (Kv3.3) in the control of the presynaptic AP spike waveforms. Importantly, mice that lack Kv3.3 showed auditory response deficits, including increases in pre-synaptic AP halfwidth, AP latency, AP jitter and spontaneous activity. The in vivo recordings are impressive. The study contains an extensive data set and makes a compelling argument for the uniquely important role for Kv3.3 in synaptic transmission. Overall, the findings will significantly advance our understanding of the calyx of Held and the neural circuit of sound processing, as well as the functions of Kv3 channels.
(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 #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study the authors make a compelling case for Kv3.3 in the control of the presynaptic AP waveform at the calyx of Held within the mouse auditory brainstem. The authors previously showed a predominant expression of Kv3.1 and Kv3.3, but not Kv3.2 or Kv3.4, in the MNTB via mRNA in situ studies and postsynaptic recordings. The authors now show that deletion of Kv3.3, but not Kv3.1, in knockout mice cause a broadening in the immature presynaptic AP waveform. This broadening presumably leads to more calcium influx to the presynaptic terminal (although not shown), which increases the peak and charge of a post-synaptic AMPA-mediated EPSC. These broad presynaptic APs also correlated with faster rates of short-term depression, but faster recovery from depression, presumably through calcium-dependent mechanisms …
Reviewer #1 (Public Review):
In this study the authors make a compelling case for Kv3.3 in the control of the presynaptic AP waveform at the calyx of Held within the mouse auditory brainstem. The authors previously showed a predominant expression of Kv3.1 and Kv3.3, but not Kv3.2 or Kv3.4, in the MNTB via mRNA in situ studies and postsynaptic recordings. The authors now show that deletion of Kv3.3, but not Kv3.1, in knockout mice cause a broadening in the immature presynaptic AP waveform. This broadening presumably leads to more calcium influx to the presynaptic terminal (although not shown), which increases the peak and charge of a post-synaptic AMPA-mediated EPSC. These broad presynaptic APs also correlated with faster rates of short-term depression, but faster recovery from depression, presumably through calcium-dependent mechanisms of synaptic release and vesicle recovery, respectively. In MNTB neurons, while Kv3.3KO mice were unable to maintain to steady state firing during 600Hz stimulation of the synaptic inputs, Kv3.1KO mice developed a depolarized plateau that took longer to recover to baseline membrane potentials compared to WT or Kv3.3KO mice. Importantly, Kv3.3 mice showed auditory response deficits, including increases in pre-synaptic AP halfwidth, synaptic delay, MNTB AP halfwidth, AP latency, AP jitter and spontaneous activity. These in vivo recordings are impressive. Overall, the study contains an extensive data set and makes a compelling argument for the uniquely important role for Kv3.3 in presynaptic transmission at the Calyx of Held synapse.
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Reviewer #2 (Public Review):
In this manuscript, the authors tested specific roles of Kv3.1 and Kv3.3 channels at the calyx of Held presynaptic terminal, by combining global knockout (KO) mice, channel blocker, in vitro slice recording and in vivo extracellular recording. They found that Kv3.3 deletion but not Kv3.1 deletion increased presynaptic AP duration, postsynaptic EPSC amplitude, and short-term depression of EPSCs during high frequency transmission. Most interestingly, using in vivo extracellular recording in MNTB, the authors found that the neuronal response to sound in MNTB was delayed in the Kv3.3 KO mice, with higher spontaneous and lower evoked short-term firing, thereby reducing signal-to-noise ratio. Using computational modeling, the authors further showed that Kv3.3 deletion enhanced EPSC and short-term depression, …
Reviewer #2 (Public Review):
In this manuscript, the authors tested specific roles of Kv3.1 and Kv3.3 channels at the calyx of Held presynaptic terminal, by combining global knockout (KO) mice, channel blocker, in vitro slice recording and in vivo extracellular recording. They found that Kv3.3 deletion but not Kv3.1 deletion increased presynaptic AP duration, postsynaptic EPSC amplitude, and short-term depression of EPSCs during high frequency transmission. Most interestingly, using in vivo extracellular recording in MNTB, the authors found that the neuronal response to sound in MNTB was delayed in the Kv3.3 KO mice, with higher spontaneous and lower evoked short-term firing, thereby reducing signal-to-noise ratio. Using computational modeling, the authors further showed that Kv3.3 deletion enhanced EPSC and short-term depression, indicating increased vesicle release probability and accelerated activity-dependent vesicle replenishment. Taken together, the authors concluded that Kv3.3 but not Kv3.1 enables short duration and temporal precision of APs to maintain transmission at high frequencies and during sustained synaptic activity.
Strengths:
(1) Combine Kv3.1 and Kv3.3 global KOs with 1 mM TEA to dissect differential roles of the two channel subunits in regulating presynaptic APs and postsynaptic EPSCs.
(2) In vivo recording of MNTB neuronal activities in response to sound in KO mice is highly interesting.
(3) Overall, a substantial amount of electrophysiology results were included in this manuscript. It is technically solid.Weaknesses:
(1) In vitro slice recording was performed in mouse pups (P10-P25), whereas in vivo recording was performed in older mice (6 months). It is not clear why the experiments were done in mice with such huge age difference. The synapse might not be fully mature around P10-P25. Kv channels also tend to have increased expression during development. Were there any published results of immuno-electron microscopy to demonstrate the presence and expression levels of Kv3.3 and Kv3.1 channels in the calyx of Held terminals during these developmental stages?
(2) There is potential compensatory effect from other Kv3 subunits in Kv3.1 or Kv3.3 global KO mice. Potential developmental changes caused by global KO should be more extensively discussed.
(3) In Figure 2, mini EPSCs should also be analyzed to help dissecting the role of Kv3.3 at different stages of synaptic transmission.
(4) Simultaneous pre- and post-synaptic recording will be challenging, but can more directly dissect the roles of Kv3.3 in synaptic transmission.
(5) The potential effect of anaesthesia on in vivo recording in response to sound should be discussed. -
Reviewer #3 (Public Review):
In this paper, the authors have used Kv3.1 and Kv3.3 KO mice and examined the roles of presynaptic firing. K channels are important in regulating presynaptic excitability and pharmacological dissection has been done by Takahashi and Forsyjte groups, but this study is novel because they have used KO mice. They used pre- and postsynaptic recording and in vivo physiology.
In Kv3.1 mice, the phenotype was not so strong. In Kv3.3 KO mice, they have found broader action potentials (AP) . In addition, they found increased EPSC amplitudes possibly due to broader APs. They also found deeper synaptic depression during repetitive activity, and increased recovery from synaptic depression, consistent with pharmacological study by Wang and Kaczmarek.
They also examined the effects on postsynaptic firing but have complex …
Reviewer #3 (Public Review):
In this paper, the authors have used Kv3.1 and Kv3.3 KO mice and examined the roles of presynaptic firing. K channels are important in regulating presynaptic excitability and pharmacological dissection has been done by Takahashi and Forsyjte groups, but this study is novel because they have used KO mice. They used pre- and postsynaptic recording and in vivo physiology.
In Kv3.1 mice, the phenotype was not so strong. In Kv3.3 KO mice, they have found broader action potentials (AP) . In addition, they found increased EPSC amplitudes possibly due to broader APs. They also found deeper synaptic depression during repetitive activity, and increased recovery from synaptic depression, consistent with pharmacological study by Wang and Kaczmarek.
They also examined the effects on postsynaptic firing but have complex effects. In Kv3.3 KO mice, postsynaptic firing loses fidelity especially during the stimulus train to the presynaptic terminal. Although it is interesting, the effects can be due to presynaptic and postsynaptic. Kv3.3 channels are expressed in the postsynaptic cell. In addition, sound-evoked postsynaptic firing in vivo is more complex, because loss of Kv3.3 affects not only the calyx of Held synapse, but others, such as hair cells, auditory nerve fibers and cochlear nucleus. These points may be carefully discussed.
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