Comparative Biochemistry and Physiology Part A 132 (2002) 477–487 1095-6433/02/$ - see front matter  2002 Elsevier Science Inc. All rights reserved. PII:S1095-6433 Ž 02 . 00093-4 Temperature and pHy CO modulate respiratory activity in the 2 isolated brainstem of the bullfrog (Rana catesbeiana) Rey D. Morales, Michael S. Hedrick* Department of Biological Sciences, California State University, Hayward, Hayward, CA 94542, USA Received 17 August 2001; received in revised form 21 February 2002; accepted 3 March 2002 Abstract The effects of temperature and pHyCO were examined in isolated brainstem preparations from adult North American 2 bullfrogs (Rana catesbeiana). These experiments were undertaken to determine the effects of temperature on fictive breathing, central pHyCO chemoreception, and to examine potential alphastat regulation of respiration in vitro. Adult 2 bullfrog brainstem preparations were isolated, superfused with an artificial cerebrospinal fluid (aCSF) and respiratory- related neural activity was recorded from cranial nerves V, X and XII. In Series I experiments (Ns8), brainstem preparations were superfused with aCSF equilibrated with 2% CO at temperatures ranging from 10 to 30 8C. Neural 2 activity was present in all preparations in the temperature range of 15–25 8C, but was absent in most preparations when aCSF was at 10 or 30 8C. The absence of fictive breathing at high (30 8C) temperatures was transient since fictive breathing could be restored upon returning the preparation to 20 8C. In Series II experiments (Ns10), preparations were superfused with aCSF equilibrated with 0%, 2% and 5% CO at temperatures of 15, 20 and 25 8C. Fictive breathing 2 frequency ( f ) was significantly dependent upon aCSF pH at all three temperatures, with slopes ranging from y0.82 R min pH unit (15 8C) to y3.3 min pH unit (20 8C). There was a significant difference in these slopes (P- y1 y1 y1 y1 0.02), indicating that central chemoreceptor sensitivity increased over this temperature range. Fictive breathing frequency was significantly dependent upon the calculated alpha-imidazole (a ) ionization (P-0.05), consistent with the alphastat Im hypothesis for the effects of temperature on the regulation of ventilation. However, most of the variation in f was not R explained by a (R s0.05), suggesting that other factors account for the regulation of fictive breathing in this 2 Im preparation. The results indicate that the in vitro brainstem preparation of adult bullfrogs has a limited temperature range (15–25 8C) over which fictive breathing is consistently active. Although there is a close correspondence of ventilation in vitro and in vivo at low temperatures, these data suggest that, as temperature increases, changes in ventilation in the intact animal are likely to be more dependent upon peripheral feedback which assumes a greater integrative role with respect to chemoreceptor drive, respiratory frequency and tidal volume.  2002 Elsevier Science Inc. All rights reserved. Keywords: Central pattern generator; Fictive breathing; Frog; Central chemoreceptors; Temperature; Carbon dioxide; pH; Alphastat regulation 1. Introduction Breathing in amphibians and other vertebrates is a behavioral motor activity that originates with a neural network localized to the medulla (Taylor et al., 1999; Wang et al., 1999). The neural *Corresponding author. Tel.: q1-510-885-7443; fax: q1- 510-885-4747. E-mail address: mhedrick@csuhayward.edu (M.S. Hedrick). network, or central pattern generator (CPG), that provides motoneuronal output to respiratory mus- cles has not been clearly delineated in ectothermic vertebrates; however, in mammals, the pre-Botzin- ¨ ger Complex is hypothesized to be the primary rhythm-generating region within the medulla (Smith et al., 1991; Gray et al., 2001). In recent years, examination of the respiratory CPG from a variety of vertebrates has made use of in vitro brainstem preparations that have rendered the prob-