Activation of respiratory muscles does not occur during cold-submergence in bullfrogs,Lithobates catesbeianus

Abstract

Semiaquatic frogs may not breathe air for several months because they overwinter in ice-covered ponds. In contrast to many vertebrates that experience decreased motor performance after inactivity, bullfrogs, Lithobates catesbeianus, retain functional respiratory motor processes following cold-submergence. Unlike mammalian hibernators with unloaded limb muscles and inactive locomotor systems, respiratory mechanics of frogs counterintuitively allow for ventilatory maneuvers when submerged. Thus, we hypothesized that bullfrogs generate respiratory motor patterns during coldsubmergence to avoid disuse and preserve motor performance. Accordingly, we measured activity of respiratory muscles (buccal floor compressor and glottal dilator) via electromyography in freely behaving bullfrogs at 20 and 2°C. Although we confirm that ventilation cycles occur underwater at 20°C, bullfrogs did not activate either respiratory muscle when submerged acutely or chronically at 2°C. We conclude that cold-submerged bullfrogs endure respiratory motor inactivity, implying that other mechanisms, excluding underwater muscle activation, maintain a functional respiratory motor system throughout overwintering.

Synaptic up-scaling preserves motor circuit output after chronic, natural inactivity

“…American bullfrogs often overwinter in ice-covered ponds with no need to use their lungs for breathing during cold winter months due to adequate skin gas exchange at low temperatures ( Tattersall and Ultsch, 2008 ). Although frogs retain a relatively high locomotor capacity during overwintering submergence ( Tattersall and Boutilier, 1999 ), important muscles of the respiratory apparatus, buccal floor constrictors and glottal dilators, are inactive during cold-submergence ( Santin and Hartzler, 2017 ). Despite months of respiratory motor inactivity, upon forced emergence at warm temperature bullfrogs immediately exhibit breathing movements, ventilate to match resting metabolic demands, increase ventilation during exposure to low oxygen, and generate respiratory motor output from the brainstem ( Santin and Hartzler, 2016a ; Santin and Hartzler, 2016b ).…”

“…Here, we take advantage of an adult animal (American bullfrogs, Lithobates catesbeianus ) that normally undergoes drastic and prolonged reductions in neuronal activity to understand if compensatory mechanisms commonly evoked during artificial deprivation of neuronal firing occur in an environmentally relevant setting. We demonstrate that a well-described mechanism of homeostatic plasticity, up-scaling of excitatory synapses, occurs in motoneurons innervating a primary respiratory muscle from bullfrogs after 2 months in a submerged-aquatic, overwintering habitat: a natural environment that induces complete respiratory motor inactivity ( Santin and Hartzler, 2017 ). Strikingly, we further identify that increased excitatory synaptic strength onto these motoneurons enhances population motoneuron output from the respiratory network, thereby acting to preserve respiratory motor drive to critical respiratory muscles under conditions when breathing would be obligatory at warm temperatures after 2 months without breathing movements ( Santin and Hartzler, 2016a ).…”

Motor inactivity in hibernating frogs: Linking plasticity that stabilizes neuronal function to behavior in the natural environment

“…This environment, therefore, leaves the motor system that drives ventilation silent for several months during hibernation. This has been verified by electromyography (EMG) recording; when frogs are chronically submerged in cold water, neuromuscular activity associated with breathing movements is absent (Figure 1a) (Santin & Hartzler, ). The hibernation environment, therefore, induces a chronic and massive activity challenge, which in many other systems, has been shown to trigger compensatory or homeostatic mechanisms.…”

“…(b) Ventilatory airflow traces at about 22°C in a frog before overwintering (left panel) and a different frog immediately after overwintering (right panel). Raw traces in A–B were part of the data sets from Santin and Hartzler () and Santin and Hartzler (), but these raw traces were not shown in the original studies. Large amplitude events represent airflow associated with lung breathing.…”

“…Since EMG findings suggested that motoneurons are silent throughout the winter months, respiratory motoneurons were likely candidates to observe mechanisms of homeostatic plasticity that directly cause behavior in the animal. We studied motor neurons of Cranial Nerve X (CN X) that innervate the laryngeal dilator, a respiratory muscle in anuran amphibians (Sakakibara, ) which is also silent during hibernation (Santin & Hartzler, ). We found that these neurons mount a well‐established compensatory response to the hibernation environment using a mechanism known as “synaptic scaling.” Synaptic scaling has long been recognized as a homeostatic process that “scales” all postsynaptic AMPA glutamate receptors up or down by the same relative amount in the opposite direction of an activity challenge (Desai, Cudmore, Nelson, & Turrigiano, ; Gonzalez‐Islas & Wenner, ; Knogler, Liao, & Drapeau, ; Turrigiano et al, ).…”