Hypoxia switches episodic breathing to singlet breathing in red-eared slider turtles (Trachemys scripta) via a tropisetron-sensitive mechanism

Highlights

• Hypoxia-induced changes in turtle breathing were examined.

• Hypoxia increased singlet breathing and decreased episodic breathing.

• Hypoxia-induced singlet breathing is abolished by tropisetron administration.

Abstract

Hypoxia-induced changes in the chelonian breathing pattern are poorly understood. Thus, breathing was measured in freely swimming adult red-eared slider turtles breathing air prior to breathing nitrogen for 4 h. Ventilation increased 10-fold within 10 min due to increased breath frequency and tidal volume. Breaths/episode decreased by ∼50% within after 1 h of hypoxia while the number of singlet breaths increased from 3.1 ± 1.6 singlets/h to a maximum of 66.1 ± 23.5 singlets/h. Expiratory and inspiratory duration increased during hypoxia. For doublet and triplet breaths, expiratory duration increased during the first breath only, while inspiratory duration increased for all breaths. Tropisetron (5-HT₃ receptor antagonist, 5 mg/kg) administration prior to hypoxia attenuated the hypoxia-induced increase in singlet breath frequency. Along with results from previous in vitro studies, this study suggests that 5-HT₃ receptor activation may be required for the hypoxia-induced increase in singlet breathing pattern in red-eared slider turtles.

Introduction

The vertebrate respiratory control system responds to hypoxia depending on the degree and duration of hypoxia, tolerance to hypoxia, and environmental conditions in a species-dependent manner. Under controlled experimental conditions, the acute hypoxic ventilatory response is well characterized with several distinct phases in mammals (Powell et al., 1998) and birds (Mitchell et al., 2001). For ectothermic vertebrates, however, there is less consistency in the length, duration and intensity of the hypoxic exposures, and ambient temperature (Porteus et al., 2011). Also, hypoxia in ectothermic vertebrates can cause metabolic depression (e.g., Hicks and Wang, 1999), decrease arterial PCO₂ (e.g., Davies and Sexton, 1987), and increase cardiovascular shunting of blood away from the lungs (Hicks and Wang, 1998), all of which confounds interpretation of experimental results. Thus, in ectothermic vertebrates, the hypoxic ventilatory response and its underlying physiological mechanisms are not well characterized.

In chelonians, acute hypoxia increases ventilation, but considerable variation is observed with respect to changes in breath frequency and tidal volume. For example, most studies on semi-aquatic turtles show that hypoxia increases both breath frequency and tidal volume (Glass et al., 1983, Herman and Smatresk, 1999, Hitzig and Nattie, 1982, Jackson, 1973, Wasser and Jackson, 1988). However, other studies show that hypoxia increases breath frequency with minimal or no changes in tidal volume in semi-aquatic turtles (Davies and Sexton, 1987, Vitalis and Milsom, 1986) and aquatic turtles (Burggren et al., 1977). In contrast, snapping turtles exposed to 10% oxygen increase ventilation by decreasing the non-ventilatory period, but there is no correlation between arterial oxygen levels and breath frequency (West et al., 1989). Thus, there is wide variability in hypoxia-induced changes in chelonian breathing.

In addition to altering breath frequency and tidal volume, hypoxia alters breathing pattern by decreasing the number of breaths/episode (defined as “episodicity”) in chelonians. For example, in unidirectionally-ventilated semi-aquatic turtles, the number of breaths/episode decreases by ∼50% when oxygen administration is switched from 30% to 0% oxygen (Herman and Smatresk, 1999). Likewise, in snapping turtles, exposure to 5% oxygen decreases breathing episodicity from 4.5 to 1.1 breaths/episode (Frische et al., 2000). Hypoxia-induced changes in breathing episodicity have not been systematically characterized, nor has the potential underlying mechanism been tested. Recently, we showed that episodicity in the fictive breathing pattern produced by isolated turtle brainstems can be altered by application of serotonin 5-HT₃ receptor agonist and antagonist drugs (Bartman et al., 2010). Isolated turtle brainstems in vitro are advantageous because they produce inspiratory- and expiratory-related motor output that is qualitatively similar to that produced by intact turtles, including episodic breathing (Johnson and Mitchell, 1998). Bath-applied 5-HT₃ receptor agonist drugs switch respiratory motor bursts to a singlet pattern, while 5-HT₃ receptor antagonist drugs increase the number of bursts/episode (Bartman et al., 2010). Thus, we postulate that hypoxia may decrease episodicity in intact semi-aquatic turtles by activation of central 5-HT₃ receptors.

Finally, hypoxia transforms the shape and duration of individual breaths. For example, severe hypoxia in mammals transforms phrenic inspiratory-related motor bursts of neural activity from a slowly-incrementing/rapidly-decrementing pattern associated with eupnea to a rapidly-incrementing/slowly-decrementing pattern associated with gasping in intact animals (reviewed in St John, 1996) and under in vitro conditions (Hill et al., 2011, Lieske et al., 2000). However, very little is known about hypoxia-induced changes in chelonian breathing with respect to breath duration during expiration or inspiration. In semi-aquatic turtles, expiratory and inspiratory duration increases during hypoxia, but this was attributed to the fact that more time is required for expiration and inspiration when tidal volume is increased (Glass et al., 1983). Thus, the question as to how hypoxia changes the duration of chelonian expiratory and inspiratory phases is not known.

To address these questions, breathing in awake, freely swimming adult red-eared slider turtles (Trachemys scripta) was measured by placing turtles in a water-filled tank with a small inverted air-filled chamber for breathing. A pneumotachograph was used to measure ventilation, breathing frequency, tidal volume, breaths/episode, and single breaths (“singlets”). Turtles were allowed to breathe room air before switching to nitrogen gas to induce hypoxia. Specifically, we tested whether: (1) hypoxia augmented breath frequency and tidal volume; (2) hypoxia decreased the number of breaths/episode and increased singlet breath frequency; (3) hypoxia augmented inspiratory duration similar to mammals or altered expiratory duration; and (4) hypoxia-induced decrease in episodicity was altered by serotonin 5-HT₃ receptor antagonist drug adminstration. Preliminary data were published in abstract form (Johnson, 2010).