Motor cortex analogue neurons in songbirds utilize Kv3 channels to generate ultranarrow spikes
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eLife Assessment:
Zemel and colleagues provide a report on the fundamental electrophysiological properties of motor neurons driving song in the zebrafish and provide complementary information about cell morphology, pharmacological sensitivity, and ion channel expression and heterogeneity. They provide mainly convincing data supporting the claim of a particular ion channel class, Kv3, that plays an important role in fast electrical spiking (action potentials) in song-related neurons.
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Abstract
Complex motor skills in vertebrates require specialized upper motor neurons with precise action potential (AP) firing. To examine how diverse populations of upper motor neurons subserve distinct functions and the specific repertoire of ion channels involved, we conducted a thorough study of the excitability of upper motor neurons controlling somatic motor function in the zebra finch. We found that robustus arcopallialis projection neurons (RAPNs), key command neurons for song production, exhibit ultranarrow spikes and higher firing rates compared to neurons controlling non-vocal somatic motor functions (dorsal intermediate arcopallium [AId] neurons). Pharmacological and molecular data indicate that this striking difference is associated with the higher expression in RAPNs of high threshold, fast-activating voltage-gated Kv3 channels, that likely contain Kv3.1 ( KCNC1 ) subunits. The spike waveform and Kv3.1 expression in RAPNs mirror properties of Betz cells, specialized upper motor neurons involved in fine digit control in humans and other primates but absent in rodents. Our study thus provides evidence that songbirds and primates have convergently evolved the use of Kv3.1 to ensure precise, rapid AP firing in upper motor neurons controlling fast and complex motor skills.
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eLife Assessment:
Zemel and colleagues provide a report on the fundamental electrophysiological properties of motor neurons driving song in the zebrafish and provide complementary information about cell morphology, pharmacological sensitivity, and ion channel expression and heterogeneity. They provide mainly convincing data supporting the claim of a particular ion channel class, Kv3, that plays an important role in fast electrical spiking (action potentials) in song-related neurons.
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Reviewer #1 (Public Review):
This manuscript presents evidence for Kv3 subunits being involved in shaping fast action potentials (APs) within the high-precision circuitry of the zebra finch song circuitry. The authors compare and contrast the morphology of Robustus Arcopallialis (RA) neurons with those in the adjacent intermediate arcopallium (AId) and compare their passive properties, action potential waveforms, and voltage-gated outward currents. Data using pharmacological agents known to interact with Kv3 channels reinforce their other observations.
Strengths:
1. Interesting avian model of cortical molecular mechanisms.
2. Comparative study at the level of cortical motoneurons showing those involved in fine motor control for vocalizations express high levels of Kv3.1.
3. Makes a case for convergent evolutionary utilization of Kv3.1 …Reviewer #1 (Public Review):
This manuscript presents evidence for Kv3 subunits being involved in shaping fast action potentials (APs) within the high-precision circuitry of the zebra finch song circuitry. The authors compare and contrast the morphology of Robustus Arcopallialis (RA) neurons with those in the adjacent intermediate arcopallium (AId) and compare their passive properties, action potential waveforms, and voltage-gated outward currents. Data using pharmacological agents known to interact with Kv3 channels reinforce their other observations.
Strengths:
1. Interesting avian model of cortical molecular mechanisms.
2. Comparative study at the level of cortical motoneurons showing those involved in fine motor control for vocalizations express high levels of Kv3.1.
3. Makes a case for convergent evolutionary utilization of Kv3.1 supporting fast spiking.
4. Clearly shows other Kv3 subunits are present in the nuclei under study.
5. Employs well-characterised pharmacological tools to support the physiology.Weaknesses:
1. Comparison with Betz Cells comes across as of secondary importance and is perhaps a discussion point rather than the first introductory paragraph.
2. Fails to adequately quantify the absolute levels of Kv3 mRNA or protein in the zebra finch brain nuclei.
3. The comparison of % or fold differences between the two avian nuclei (RA and AId neuron) masks important quantitative evidence and the contribution of multiple subunits to functional channels is not well developed.
4. The voltage-clamp data suggests that the large TEA-sensitive current is too slow to dominantly contribute to the repolarization of a single AP (but would require sustained or cumulative depolarization to be activated), while the fast transient current which could contribute to single APs, is not sufficiently characterised.
5. It is not possible to conclude that the pharmacology is specific for Kv3.1, it is at best indicative, and the absence of more precise molecular tools (e.g. knockout or gene-edited animals) undermines the authors' justification of the zebra finch as an accessible model.
6. Although the authors acknowledge the presence of other Kv3 subunits, the report fails to explain whether they are functional, but focuses on Kv3.1 as being dominant, without sufficiently addressing how other subunits contribute (perhaps as heteromeric assemblies of subunits). -
Reviewer #2 (Public Review):
The mechanisms of action potential firing were studied by whole-cell patch-clamp recordings in acute brain slices of the zebra finch. The study builds on the initial finding by Zemel et al. (2021) that the action potentials of robustus arcopallialis projection neurons (RAPNs) have an exceptional small half-duration of about 0.2 ms at 40C. The authors, therefore, set out to investigate the mechanisms of action potential repolarization. They use an impressive set of complementary techniques including voltage clamp and current clamp recordings, pharmacological interventions with classical and novel subunit-specific blockers, in situ hybridization, and comparative genomics of the KCNC/Kv3 potassium channel genes. The data convincingly demonstrate that the Kv3.1 but not Kv3.2-Kv3.4 nor Kv1.1/1.2/1.6, Kv7, or BK …
Reviewer #2 (Public Review):
The mechanisms of action potential firing were studied by whole-cell patch-clamp recordings in acute brain slices of the zebra finch. The study builds on the initial finding by Zemel et al. (2021) that the action potentials of robustus arcopallialis projection neurons (RAPNs) have an exceptional small half-duration of about 0.2 ms at 40C. The authors, therefore, set out to investigate the mechanisms of action potential repolarization. They use an impressive set of complementary techniques including voltage clamp and current clamp recordings, pharmacological interventions with classical and novel subunit-specific blockers, in situ hybridization, and comparative genomics of the KCNC/Kv3 potassium channel genes. The data convincingly demonstrate that the Kv3.1 but not Kv3.2-Kv3.4 nor Kv1.1/1.2/1.6, Kv7, or BK channels mediate the rapid repolarization. The manuscript is clearly written and the data and the presentation of the data are of the highest scientific quality. The study is of interest to a broad readership because the zebra finch is a fascinating and novel model to investigate the mechanisms of rapid motor control. The similarities of these neurons of the zebra finch with the specialized Betz cells in the motor cortex of humans and other primates demonstrates the exciting advantages of this animal model in comparison with well-established rodent models to investigate the mechanisms of complex sensory-motor control in vertebrates.
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Reviewer #3 (Public Review):
This work describes intracellular recordings from motor neurons of the zebrafinch. The authors use isolated brain slices allowing careful analysis of both voltage- and current-clamp recordings to document differences in action potentials in two motor cortical areas. RAPN neurons are associated with vocal commands that generate bird song, while Ald neurons are also motor neurons, but are not involved in song.
RAPN neurons are found to have much faster action potentials than Ald neurons, and pharmacological experiments provide evidence for the involvement of a particular class of voltage-gated potassium channels, Kv3, in RAPNs that presumably contributes to a faster rate of action potential repolarization and a concomitant narrowing of the action potential width. A set of experiments is included to verify that …
Reviewer #3 (Public Review):
This work describes intracellular recordings from motor neurons of the zebrafinch. The authors use isolated brain slices allowing careful analysis of both voltage- and current-clamp recordings to document differences in action potentials in two motor cortical areas. RAPN neurons are associated with vocal commands that generate bird song, while Ald neurons are also motor neurons, but are not involved in song.
RAPN neurons are found to have much faster action potentials than Ald neurons, and pharmacological experiments provide evidence for the involvement of a particular class of voltage-gated potassium channels, Kv3, in RAPNs that presumably contributes to a faster rate of action potential repolarization and a concomitant narrowing of the action potential width. A set of experiments is included to verify that the findings obtained under normal ex vivo recording conditions (23 deg C) are retained under more physiological conditions (40 deg C). Consistent with the role of Kv3, the action potentials, and underlying potassium currents, are modified by imperfect pharmacological tools TEA, 4AP, and AUT5. Even though imperfect, together they provide support for the role of Kv3. Examination of transcripts for Kv3 family members documents that Kv3.1 is more highly expressed in RAPN than Ald neurons.
The experiments are adequately replicated and the paper is written very clearly. The authors claim that these cells are similar to Betz cells, highly specialized pyramidal neurons mainly found in the primate motor cortex and that they may play a similar role in primates and birds in generating fine motor behavior.
Some weaknesses include missing controls such as reversibility of pharmacological effects and improved statistical analysis. In addition, the linkage of RAPN neurons to Betz cells is not very strong.
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