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First published online June 11, 2007
Journal of Experimental Biology 210, 2046-2056 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003202

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Effects of medullary Raphé stimulation on fictive lung ventilation during development in Rana catesbeiana

Olivier Belzile¹, Roumiana Gulemetova² and Richard Kinkead^2,*

¹ Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039, USA
² Department of Pediatrics, Laval University, Centre de Recherche Hôpital St-François d'Assise, Québec City, Québec, G1L 3L5, Canada

View larger version (20K): [in this window] [in a new window]   Fig. 1. (A) Schematic representation of bullfrog brainstem showing the Raphé stimulation site. (B–D) Integrated (top trace) and raw (bottom trace) trigeminal neurograms from a pre-metamorphic tadpole, (B) under baseline condition, (C) during and after electrical stimulation of the medullary Raphé with drug-free artificial cerebrospinal fluid (aCSF) and (D) in the presence of methiothepin in aCSF (50 µmol l–1). (E) The changes in respiratory-related motor output after a 20-min `wash out' period with drug-free aCSF.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Trigeminal neurograms comparing responsiveness to electrical stimulation of medullary Raphé neurons before (A,C,E) and during (B,D,F) bath application of the selective 5-HT3 receptor antagonist tropisetron. Recordings were obtained from brainstem preparations from pre-metamorphic tadpoles.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effects of electrical stimulation of medullary Raphé neurons on fictive lung ventilation frequency. (A,C) Responses were measured in brainstem preparations from (A) pre-metamorphic tadpoles, (B) metamorphic tadpoles, and (C) adult bullfrogs under control conditions [artificial cerebrospinal fluid (aCSF) only; open diamonds] and in the presence of a 5-HT3 receptor antagonist (tropisetron; closed circles). Note that the tropisetron concentration required to affect the frequency response (when present) decreases during development. (D,E) Stage-dependent lung burst frequency responsiveness to medullary Raphé stimulation expressed as the percentage change from baseline of the frequency response following 20 Hz stimulation (D), and as percentage change from baseline expressed as the percentage of the maximum lung burst frequency produced by each preparation (E). The black bars represent the control response (aCSF) and the grey bars represent the response in the presence of the 5-HT3 antagonist (when necessary). Values are expressed as means ± s.e.m. Asterisks indicate values statistically different from baseline values (no stimulation) at *P<0.05 and *P<0.01; indicates a value significantly different from control at P<0.01.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Changes in respiratory motor output during microinjection in the medullary Raphé. (A,B) Trigeminal neurograms showing the effects of glutamate microinjections in the medullary Raphé on fictive breathing (A) in the presence of artificial cerebrospinal fluid (aCSF) only and (B) in the presence of the selective 5-HT3 receptor antagonist tropisetron. (C,D) Control experiments consisted of measuring changes in trigeminal activity following (C) glutamate microinjection (1 nl, 0.5 mol l–1) 350 µm lateral to the medullary Raphé or (D) aCSF injection into the medullary Raphé. This figure also shows various types of burst activity observed following glutamate microinjection. Note that only bursts with a duration and amplitude similar to those observed prior to injections were counted as respiratory related (black bars). Bursts of a seizure-like activity or that were too long were not counted (grey bars).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effects of glutamate microinjections in the medullary Raphé on fictive lung ventilation frequency. (A-C) Responses were measured in brainstem preparations from (A) pre-metamorphic tadpoles, (B) metamorphic tadpoles, and (C) adult bullfrogs under control conditions [artificial cerebrospinal fluid (aCSF) only; open diamonds)] and in the presence of a 5-HT3 receptor antagonist (Tropisetron; closed circle). Note that the tropisetron concentration required to affect the frequency response (when present) decreased during development. (D,E) A between stage-group comparison was performed by expressing the frequency response following glutamate microinjection (1 nl pre- and metamorphic tadpoles; 5 nl adult frogs) as (D) the percentage change from baseline, and (E) the percentage change from baseline expressed as the percentage of the maximum lung burst frequency produced by each preparation. The black bars represent the control response (aCSF); the grey bars represent the response in the presence of the 5-HT3 antagonist (when necessary). Values are expressed as means ± s.e.m. Asterisks indicate values statistically different from baseline values (no stimulation) at *P<0.05 and **P<0.01.

View larger version (58K): [in this window] [in a new window]   Fig. 6. (A) Schematic representation of a sagittal section of the bullfrog brainstem showing the site where the rostral medullary Raphé was stimulated and level where analysis of the 5-HT immunoreactivity was performed. (B) Representative photomicrographs of cross sections comparing 5-HT immunoreactivity in the medullary Raphé (level of cranial nerve VII) of each stage group. Scale bar, 100 µm. (C) Box plots showing the number of 5-HT-immunoreactive neurons counted within the rostral medullary Raphé in the region where the stimulations were performed. The bottom of the box shows the 25th percentile and the top shows the 75th percentile. The line within the box shows the median. * Indicates a value statistically different from pre-metamorphic tadpoles; indicates a value statistically different from the metamorphic data at P<0.05.