Development of the respiratory response to hypoxia in the isolated brainstem of the bullfrogRana catesbeiana

Abstract

SUMMARY The aim of this study was to examine the effects of cellular hypoxia, and the contribution of anaerobic metabolism, on respiratory activity in bullfrogs at different stages of development. Respiratory-related neural activity was recorded from cranial nerve rootlets in isolated brainstem preparations from pre-metamorphic (Taylor–Köllros (T-K) stages VIII-XVI) and postmetamorphic tadpoles (T-K stages XXIV-XXV) and adults. Changes in fictive gill/lung activity in brainstems from pre-metamorphic tadpoles and lung activity in postmetamorphic tadpoles and adults were examined during superfusion with control (98% O2/2% CO2) or hypoxic (98%N2/2% CO2) artificial cerebrospinal fluid (aCSF). Iodoacetate (IAA; 100 μmol l–1) was used in conjunction with hypoxic aCSF to inhibit glycolysis. Gill burst frequency in pre-metamorphic brainstems did not change over a 3 h exposure to hypoxia and fictive lung burst frequency slowed significantly, but only after 3 h hypoxia. Blockade of glycolysis with IAA during hypoxia significantly reduced the time respiratory activity could be maintained in pre-metamorphic, but not in adult,brainstems. In brainstems from post-metamorphic tadpoles and adults, lung burst frequency became significantly more episodic within 5–15 min hypoxic exposure, but respiratory neural activity was subsequently abolished in every preparation. The cessation of fictive breathing was restored to control levels upon reoxygenation. Neither tadpole nor adult brainstems exhibited changes in neural bursts resembling `gasping' that is observed in mammalian brainstems exposed to severe hypoxia. There was also a significant increase in the frequency of `non-respiratory' bursts in hypoxic postmetamorphic and adult brainstems, but not in pre-metamorphic brainstems. These results indicate that pre-metamorphic tadpoles are capable of maintaining respiratory activity for 3 h or more during severe hypoxia and rely to a great extent upon anaerobic metabolism to maintain respiratory motor output. Upon metamorphosis, however, hypoxia results in significant changes in respiratory frequency and pattern, including increased lung burst episodes,non-ventilatory bursts and a reversible cessation of respiratory activity. Adults have little or no ability to maintain respiratory activity through glycolysis but, instead, stop respiratory activity until oxygen is available. This `switch' in the respiratory response to hypoxia coincides morphologically with the loss of gills and obligate air-breathing in the postmetamorphic frog. We hypothesize that the cessation of respiratory activity in post-metamorphic tadpoles and adults is an adaptive, energy-saving response to low oxygen.

Developmental changes in central O2 chemoreflex in Rana catesbeiana: the role of noradrenergic modulation

“…chemoreflex in vitro To demonstrate that our preparations produced a hypoxic response similar to the one reported previously in this species Central O 2 chemoreflex in bullfrogs (Winmill et al, 2005), these experiments first compared the effects of acute brainstem superfusion with hypoxic aCSF on fictive breathing frequencies (both lung and buccal) between two distinct developmental stages groups: adult bullfrogs and pre-metamorphic tadpoles. For this series, the protocol began by recording respiratory-related motor output for 10·min.…”

“…Furthermore, reducing O 2 levels of the artificial cerebrospinal fluid (aCSF) superfusing in vitro brainstems preparations decreases fictive lung ventilation frequency in Rana catesbeiana (Winmill et al, 2005) and newborn rat (Brockhaus et al, 1993). As this preparation is completely devoid of peripheral (sensory) inputs, these data show that this chemoreflex is of central origin.…”

“…However, such conditions are infrequent and animals rarely encounter hypoxia levels similar to the one used in our study. Although the use of less severe hypoxia may be more physiologically relevant, we chose this level mainly to reproduce previous in vitro studies (Brockhaus et al, 1993;Winmill et al, 2005). Even so, results obtained in adult bullfrog brainstems were similar to the hypoxic responses reported in intact frogs (Rose and Drotman, 1967), newborn lambs (Dawes et al, 1983;Moore et al, 1996) and newborn rats in vitro (Brockhaus et al, 1993).…”

“…The use of a higher bicarbonate concentration in the aCSF for adult bullfrogs is common (e.g. Winmill et al, 2005) and is based on the fact that (1) during bullfrog development, the transition from water to air breathing is associated with a respiratory acidosis, which is compensated by renal HCO 3 -retention (Just et al, 1973), (2) the blood-brain barrier of frog is permeable to HCO 3 - (Wright, 1972) and (3) increasing aCSF results in a more stable respiratory activity in preparations from adult bullfrogs (Kinkead et al, 1994). The superfusate was equilibrated with a 98% O 2 /2% CO 2 gas mixture to pH·7.90±0.15 for tadpoles and pH·7.8±0.15 for adult bullfrogs (Kinkead et al, 1994;Torgerson et al, 1997).…”

“…An important aspect of the work of Winmill and colleagues (Winmill et al, 2005) is that the response to central hypoxia is stage-dependent, since the frequency change recorded from…”