NaV1.1 is essential for proprioceptive signaling and motor behaviors

  1. Cyrrus M Espino
  2. Cheyanne M Lewis
  3. Serena Ortiz
  4. Miloni S Dalal
  5. Snigdha Garlapalli
  6. Kaylee M Wells
  7. Darik A O'Neil
  8. Katherine A Wilkinson
  9. Theanne N Griffith

  • Curated by eLife

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    Evaluation Summary:

    This study provides insight into the identity of the sodium channel controlling excitability in proprioceptors. Using pharmacology, gene KO, behavior, and histology, the authors show quite convincingly that NaV1.1 in sensory neurons is essential for normal motor behavior and contributes to proprioceptor excitability. The work has interesting implications for human subjects with inherited variants of Nav1.1.

    (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

The voltage-gated sodium channel (Na V ), Na V 1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for Na V 1.1 in mammalian proprioception. RNAscope analysis and in vitro patch-clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of Na V 1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting that this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of Na V 1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other Na V isoforms is sufficient to elicit activity in response to transient stimuli, Na V 1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, Na V 1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring Na V 1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest that sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which Na V 1.1 function is compromised. Collectively, we present the first evidence that Na V 1.1 is essential for mammalian proprioceptive signaling and behaviors.

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  1. Version published to 10.7554/elife.79917 on eLife
  2. Version published to 10.1101/2022.05.05.490851v2 on bioRxiv
  3. eLife

    Author Response

    Reviewer #1 (Public Review):

    This paper tackles a very important question in somatosensory biology - the identity of the sodium channel controlling excitability in proprioceptors. While whole rainforests' worth of papers have been published on sodium channels in nociceptors, there has been a significant gap in our understanding of which NaV isoforms are at play in the large fiber proprioceptors and LTMRs. Using pharmacology, gene KO, behavior, and histology, the authors show quite convincingly that NaV1.1 in sensory neurons is essential for normal motor behavior and contributes to proprioceptor excitability. Interestingly, they find NaV1.1 is haploinsufficient. This finding is all the more exciting given the many human NaV1.1 het and homo mutants and points to future possibilities for interrogating the role of this channel in human proprioception and using human tissue (e.g. iPSCs).

    We are delighted that the Reviewer finds our results address a “very important question in the field of somatosensory biology”.

    Reviewer #2 (Public Review):

    The manuscript by [Espino et al, 2022] characterizes the role of the sodium channel Nav1.1 in DRG sensory neurons, focusing on its role in proprioceptive sensory neurons. Nav1.1 expression has previously been observed in myelinated DRG neurons (including proprioceptive muscle afferents) but its significance for proprioceptive function remains unknown. In a series of molecular and in vitro patch clamp studies (using pharmacological Nav1.1 inhibitors and activators), the authors demonstrate that all proprioceptors express Nav1.1 and that this sodium channel is required for repetitive firing in the majority of proprioceptors. A pan sensory conditional deletion of Nav1.1 leads to a loss in motor coordination, suggesting that Nav1.1 in sensory neurons is required for normal motor control. While this is a somewhat generic and slightly unsatisfying conclusion, further morphological studies and ex vivo electrophysiological recordings of functionally identified muscle spindle afferents begin to offer a more interesting take on the role of Nav1.1 in proprioceptor function. First, while proprioceptor number and spindle morphology are unchanged, it appears as if the number of synapses between muscle spindle afferents to motor neurons is reduced, perhaps suggesting that a reduction in proprioceptor excitability during development affects the formation of proprioceptive sensory-motor circuits. Second, ex vivo recordings of MS afferents indicate that the loss of Nav1.1 primarily affects the static phase of their response to increases in muscle length, suggesting a role in the regulation of proprioceptor slow adaptation response properties.

    There are two clear strengths of the manuscript. First, mutations in Nav1.1 have been shown to be associated with a number of central brain disorders, including those that lead to motor impairments. The notion that a sensory neuron restricted loss of Nav1.1 similarly leads to motor coordination defects indicates that some phenotypes that previously had been suggested to be due to a central role of Nav1.1 could in fact have a peripheral basis. A second strength is that these studies further our understanding of the molecules that regulate excitability in proprioceptors and offer a foundation for further work to tease apart the molecular underpinnings of the physiological response properties of individual proprioceptor subtypes.

    While the studies generally support the main conclusion that Nav1.1 in mammalian sensory neurons is required for normal motor behaviors, the depth of some of the analyses leaves a bit more to be desired. For example, it seems that a little more could have been done to strengthen the in vitro analyses of Nav1.1 in proprioceptors with additional controls, and by expanding this analysis to genetically identified Nav1.1 mutant (heterozygous or homozygous) proprioceptors. In addition, it feels a bit of a missed opportunity that there is no further exploration of the relationship of Nav1.1 function in the context of specific proprioceptor subtypes (even if only through discussion). In addition, the observation that a loss in Nav1.1 may cause disruptions in sensorymotor connectivity could benefit from additional analyses to support these findings.

    We thank the reviewer for identifying the strengths of our study and pointing out that these new findings will “offer a foundation for further work”. We are eager to continue this line of investigation and are currently developing new approaches in the lab that will allow us to deepen our analyses in the future.

    Reviewer #3 (Public Review):

    The authors characterize the role of voltage-gated sodium channel Nav1.1 expression in proprioceptors in the peripheral nervous system. They use genetically modified mice, pharmacological blockers, and electrophysiological methods to support their claims. Albeit it was known for a long time that Nav1.1 is expressed in the peripheral nervous system, Espino et al. here present a thorough characterization of its role in proprioception and show its importance for motor behaviour, proprioceptor function, and synaptic transmission in the spinal cord. Characterizing the sodium channel subtype's function is crucial for our understanding of the function and dysfunction of the nervous system and to potentially develop new therapeutic approaches.

    We thank the Reviewer for their comments on the importance of our work investigating sodium channel function in proprioceptors.

  4. eLife

    Evaluation Summary:

    This study provides insight into the identity of the sodium channel controlling excitability in proprioceptors. Using pharmacology, gene KO, behavior, and histology, the authors show quite convincingly that NaV1.1 in sensory neurons is essential for normal motor behavior and contributes to proprioceptor excitability. The work has interesting implications for human subjects with inherited variants of Nav1.1.

    (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.)

  5. eLife

    Reviewer #1 (Public Review):

    This paper tackles a very important question in somatosensory biology - the identity of the sodium channel controlling excitability in proprioceptors. While whole rainforests' worth of papers have been published on sodium channels in nociceptors, there has been a significant gap in our understanding of which NaV isoforms are at play in the large fiber proprioceptors and LTMRs. Using pharmacology, gene KO, behavior, and histology, the authors show quite convincingly that NaV1.1 in sensory neurons is essential for normal motor behavior and contributes to proprioceptor excitability. Interestingly, they find NaV1.1 is haploinsufficient. This finding is all the more exciting given the many human NaV1.1 het and homo mutants and points to future possibilities for interrogating the role of this channel in human proprioception and using human tissue (e.g. iPSCs).

  6. eLife

    Reviewer #2 (Public Review):

    The manuscript by Espinosa et al. characterizes the role of the sodium channel Nav1.1 in DRG sensory neurons, focusing on its role in proprioceptive sensory neurons. Nav1.1 expression has previously been observed in myelinated DRG neurons (including proprioceptive muscle afferents) but its significance for proprioceptive function remains unknown. In a series of molecular and in vitro patch clamp studies (using pharmacological Nav1.1 inhibitors and activators), the authors demonstrate that all proprioceptors express Nav1.1 and that this sodium channel is required for repetitive firing in the majority of proprioceptors. A pan sensory conditional deletion of Nav1.1 leads to a loss in motor coordination, suggesting that Nav1.1 in sensory neurons is required for normal motor control. While this is a somewhat generic and slightly unsatisfying conclusion, further morphological studies and ex vivo electrophysiological recordings of functionally identified muscle spindle afferents begin to offer a more interesting take on the role of Nav1.1 in proprioceptor function. First, while proprioceptor number and spindle morphology are unchanged, it appears as if the number of synapses between muscle spindle afferents to motor neurons is reduced, perhaps suggesting that a reduction in proprioceptor excitability during development affects the formation of proprioceptive sensory-motor circuits. Second, ex vivo recordings of MS afferents indicate that the loss of Nav1.1 primarily affects the static phase of their response to increases in muscle length, suggesting a role in the regulation of proprioceptor slow adaptation response properties.

    There are two clear strengths of the manuscript. First, mutations in Nav1.1 have been shown to be associated with a number of central brain disorders, including those that lead to motor impairments. The notion that a sensory neuron restricted loss of Nav1.1 similarly leads to motor coordination defects indicates that some phenotypes that previously had been suggested to be due to a central role of Nav1.1 could in fact have a peripheral basis. A second strength is that these studies further our understanding of the molecules that regulate excitability in proprioceptors and offer a foundation for further work to tease apart the molecular underpinnings of the physiological response properties of individual proprioceptor subtypes.

    While the studies generally support the main conclusion that Nav1.1 in mammalian sensory neurons is required for normal motor behaviors, the depth of some of the analyses leaves a bit more to be desired. For example, it seems that a little more could have been done to strengthen the in vitro analyses of Nav1.1 in proprioceptors with additional controls, and by expanding this analysis to genetically identified Nav1.1 mutant (heterozygous or homozygous) proprioceptors. In addition, it feels a bit of a missed opportunity that there is no further exploration of the relationship of Nav1.1 function in the context of specific proprioceptor subtypes (even if only through discussion). In addition, the observation that a loss in Nav1.1 may cause disruptions in sensory-motor connectivity could benefit from additional analyses to support these findings.

  7. eLife

    Reviewer #3 (Public Review):

    The authors characterize the role of voltage-gated sodium channel Nav1.1 expression in proprioceptors in the peripheral nervous system. They use genetically modified mice, pharmacological blockers, and electrophysiological methods to support their claims. Albeit it was known for a long time that Nav1.1 is expressed in the peripheral nervous system, Espino et al. here present a thorough characterization of its role in proprioception and show its importance for motor behaviour, proprioceptor function, and synaptic transmission in the spinal cord. Characterizing the sodium channel subtype's function is crucial for our understanding of the function and dysfunction of the nervous system and to potentially develop new therapeutic approaches.

  8. Version published to 10.1101/2022.05.05.490851v1 on bioRxiv