Characterization of laryngeal motor neuron properties in the American bullfrog, Lithobates catesbieanus

https://doi.org/10.1016/j.resp.2021.103745Get rights and content

Highlights

  • Motor control of the glottal dilator regulates lung airflow in anuran amphibians.

  • We characterized intrinsic membrane properties of motor neurons that mainly innervate the glottal dilator in American bullfrogs, Lithobates catesbieanus.

  • Motor neurons had highly variable passive and active properties.

  • Despite variability within the population, motor neurons could be separated into two groups using hierarchal clustering and principal component analysis.

  • The two cell phenotypes included those with fast firing rate and adaptation, and slow firing rates with little adaptation.

Abstract

Motor neurons represent the final output from the central respiratory network. American bullfrogs, Lithobates catesbieanus, have provided insight into development and plasticity of the breathing control system, yet cellular aspects of bullfrog motor neurons are not well-described. In this study, we characterized properties of laryngeal motor neurons that produce motor outflow to the glottal dilator, a muscle that gates airflow to the lungs of anurans. To this end, we measured several intrinsic membrane properties of labeled laryngeal motor neurons in brain slices. Using unsupervised clustering analyses, we identified two broad classes of motor neurons: those with high firing rates and strong adaptation (∼70 %), and those with lower firing rates and less adaptation (∼30 %). These results suggest that two neuronal cell types innervate the glottal dilator, roughly aligning with the composition of fast and slower twitch fibers of this muscle. In sum, these data reinforce the need to consider cell-type when assessing motor neuron function in the respiratory network.

Introduction

Diverse vertebrate species have produced insights into the evolution and function of the respiratory control system. Among several of these animals, American bullfrogs, Lithobates catesbieanus, continue to inform the study of how the respiratory network develops (Janes et al., 2019b), responds to environmental change (Santin, 2019), and generates rhythmic output (Baghdadwala et al., 2016). Although amphibians and mammals share many features of the respiratory network (Baghdadwala et al., 2016; Kinkead, 2009), one major difference lies in the mechanics of ventilation. In particular, mammals use the diaphragm to generate negative pressure for lung inflation, while bullfrogs and other anurans use a positive pressure system to inflate the lungs. The generation of positive pressure involves coordination among the nares, buccal floor, and glottis. At the start of a lung ventilation cycle, the nares remain open and the buccal floor dilates to draw air into the buccal cavity; the nares then close, the glottis opens, and the muscles of the buccal floor compress the buccal cavity to drive air into the lungs (Vitalis and Shelton, 1990; West and Jones, 1975). Lung breaths involve the activation of several motor nerves, including the mandibular branch of the trigeminal nerve, the sternohyoid and main branches of the hypoglossal nerve, and the laryngeal branch of the vagus nerve (Kogo et al., 1994; Sakakibara, 1983). Although bullfrogs have well-defined respiratory mechanics and motor outflows, the electrophysiological properties of motor neurons that innervate muscles involved in generating breathing have received less attention.

A characterization of motor function at the cellular level can inform the study of mechanisms that shape network output (Harris-Warrick, 2002), motor unit recruitment during respiratory and non-respiratory behaviors (Mantilla and Sieck, 2003), and neuronal plasticity (Schulz, 2006). One study from bullfrogs assessed motor neuron function of trigeminal, laryngeal, pharyngeal, and hypoglossal motor neurons and correlated their activities with different phases of the respiratory cycle in situ (Kogo and Remmers, 1994). More recently, Burton and Santin (2020) found that hypoglossal motor neurons from American bullfrogs enhance excitatory synaptic strength in response to lactate ions, acting as potential metabolite sensors controlling respiratory motor outflow. In addition, Janes et al. (2019a) assessed properties of individual motor neurons that innervate the buccal floor. They identified two types of motor neurons that could be distinguished based on the expression of the hyperpolarization-activated current, Ih. Each cell type displayed electrophysiological properties consistent with recruitment during low-force respiratory behaviors such as ventilation of the buccal cavity or more forceful contractions that occur during lung ventilation. Thus, an understanding of motor neuron diversity has the potential to provide insight into the cellular basis of different ventilatory behaviors in anurans.

We recently assessed function of laryngeal motor neurons that innervate the glottal dilator in bullfrogs before and after underwater submergence, as can occur during aquatic overwintering (Santin et al., 2017). Bullfrogs do not exhibit respiratory motor activity while submerged in cold water but breathe normally soon after emergence (Santin and Hartzler, 2016a, 2017). Thus, our recent work has sought to define plasticity that might allow for normal motor performance after such a dramatic period of inactivity that, in other species and neural systems, typically disrupts motor function. We found that motor neurons undergo increases in the amplitude of excitatory postsynaptic currents, indicative of upregulated AMPA-glutamate receptors. This type of plasticity is consistent with a compensatory mechanism termed “synaptic scaling” which we showed to enhance motor output after winter. Intrinsic excitability, as assessed by firing responses to current injections, did not appear to differ. However, we observed an approximately 10-fold range in intrinsic excitability in these experiments. Heterogeneity of excitability exists within and across motor pools, often reflecting the types of muscle fibers neurons innervate (Deardorff et al., 2013; Hadzipasic et al., 2014; Mantilla and Sieck, 2003). Thus, multiple motor neuron types may innervate the glottal dilator muscle and potentially complicate our conclusion that plasticity of intrinsic membrane properties that influence neuronal firing did not occur during winter, as plasticity of intrinsic properties often occurs in a cell-type specific manner (Schulz, 2006; Temporal et al., 2012). To better understand the variability that exists in motor neurons that form the laryngeal branch of the vagus nerve, we characterized membrane properties of a relatively large number of cells from control bullfrogs and used unsupervised clustering methods to estimate the number of cell types. To this end, we labeled laryngeal motor neurons and used whole-cell patch clamp electrophysiology to measure intrinsic properties of neurons in brainstem slices. We then used hierarchal clustering and principal component analysis to group cells based on their intrinsic properties and ran multiple analyses for cluster number selection to estimate the number of cell types in the motor pool.

Section snippets

Ethical approval

All experiments were approved by the Institutional Animal Care and Use Committee at Wright State University (protocol #1047).

Animals

14 Adult female American bullfrogs (Lithobates catesbieansus) weighting approximately 100 g were purchased from Rana Ranch (Twin Falls, ID, USA). Bullfrogs were housed in plastic tanks in aerated, dechlorinated tap water at 22 °C and fed crickets twice per week ad libitum. Frogs had access to both wet and dry areas.

Dissection, motor neuron labeling, and whole-cell patch clamp electrophysiology

Brainstem dissection, brain slices, and solutions were

Results

The purpose of this study was to characterize the electrophysiological diversity of motor neurons that innervate the glottal dilator in bullfrogs. Fig. 1A shows whole-cell current clamp recordings of labeled neurons in brainstem slices and the 13 electrophysiological properties measured. The ranges obtained for each variable are shown in Fig. 1B–D as box and whisker plots, where boxes show the interquartile range and whiskers show the 5–95 % range (n = 67). We found a wide range in variables

Discussion

Motor neurons with different electrophysiological properties often compose a motor pool in vertebrates. An understanding of cell type diversity can inform aspects of respiratory function including motor recruitment and plasticity. In motor neurons that innervate the glottal dilator from bullfrogs, we previously identified a wide range of steady-state firing rates across a small population of cells (Santin et al., 2017), suggestive of multiple neuronal types. Thus, we recorded from a relatively

Acknowledgements

This work was funded in part by grants from the National Institutes of Health (1R15NS112920-01A1 to JS), the U.S. Department of Defense (W911NF2010275 to JS), and laboratory startup funds from The University of North Carolina-Greensboro (to JS).

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