Elsevier

Brain Research

Volume 1090, Issue 1, 23 May 2006, Pages 134-145
Brain Research

Research Report
GABAergic and glycinergic inhibitory mechanisms in the lamprey respiratory control

https://doi.org/10.1016/j.brainres.2006.03.056Get rights and content

Abstract

The specific role of γ-aminobutyric acid (GABA) and glycine receptors in respiratory rhythm generation and pattern formation was investigated in in vitro brainstem preparations from adult lampreys by analyzing the changes in respiratory activity induced by bath application of specific antagonists, agonists, and uptake blockers. GABAA receptor blockade by bicuculline or picrotoxin increased both the frequency and amplitude of respiratory bursts. Similar effects were observed after glycine receptor blockade by strychnine. Combined bath application of bicuculline and strychnine markedly increased the frequency and amplitude of respiratory activity. These responses were associated, especially at the higher concentrations of the two drugs, with the appearance of tonic activity and irregular, high-frequency bursts followed by transient depression of respiratory activity. GABAA and glycine receptor agonists suppressed respiratory activity. These effects were prevented by bath application of the corresponding specific antagonists. GABAB receptor blockade by 2-hydroxysaclofen reduced the respiratory frequency but increased the peak amplitude of respiratory bursts. Activation of GABAB receptors suppressed respiratory activity. These responses were prevented by 2-hydroxysaclofen. Neither GABAC receptor agonist nor antagonist had any effects on respiration. Depression of both the frequency and amplitude of respiratory bursts was induced by blockades of GABA and glycine uptake using, respectively, nipecotic acid and sarcosine. The results suggest that GABA- and glycine-mediated inhibition is not essential for respiratory rhythm generation in the adult lamprey, although it appears to exert potent influences on respiratory activity and to have a role in maintaining a stable and regular breathing pattern.

Introduction

The neural mechanisms involved in respiratory rhythm generation have been investigated mainly in mammals (Ezure, 2004, Feldman et al., 2003); however, several issues still remain to be resolved. Further insights into the mechanisms underlying the genesis of rhythmic activities may arise from studies on lower vertebrates, such as the lamprey (see, e.g., Grillner, 2003, Marder and Calabrese, 1996). In the adult lamprey, breathing is produced by synchronous contractions of the branchial muscles that force water out of the gill openings. The subsequent refilling of the gill sacs is produced by the elastic recoil of the cartilaginous gill baskets surrounding them (Rovainen, 1977). The isolated brain spontaneously produces respiratory neuronal activity in vitro; this activity closely resembles that underlying the respiratory behaviour of intact animals and persists after transections of the brain at the obex and isthmus levels (e.g., Bongianni et al., 1999, Bongianni et al., 2002, Rovainen, 1977, Rovainen, 1983, Russell, 1986, Thompson, 1985). Thus, both the neural network responsible for respiratory rhythm generation and respiratory motoneurons are located within the brainstem. Little is known as regards the neural mechanisms underlying respiratory rhythmogenesis in the lamprey. Respiratory motoneurons are situated in the facial, glossopharyngeal and, especially, in the vagal nuclei, but the respiratory central pattern generator (CPG) has not yet been clearly identified (e.g., Bongianni et al., 1999, Bongianni et al., 2002, Guimond et al., 2003, Rovainen, 1977, Rovainen, 1983, Rovainen, 1985, Russell, 1986, Thompson, 1985 also for further references).

Recent studies in the in vitro brainstem preparation of the adult lamprey have shown that endogenously released excitatory amino acids are involved in the control of the timing and intensity components of the breathing pattern through an action on ionotropic and metabotropic glutamate receptors (Bongianni et al., 1999, Bongianni et al., 2002). More specifically, evidence has been provided as regards the fact that the respiratory activity in the lamprey is crucially dependent upon the activation of non-N-methyl-d-aspartate (non-NMDA) receptors, given that the blockade thereof completely suppresses the respiratory motor output (Bongianni et al., 1999).

On the other hand, the involvement of inhibitory amino acids in the respiratory control has recently been debated. In mammals, it has been proposed that the functional role of γ-aminobutyric acid (GABA) and glycine receptors becomes increasingly important during maturation (reviewed in Ballanyi et al., 1999, Ezure, 2004, Feldman et al., 2003, Haji et al., 2000, Richter and Spyer, 2001), although the results of some studies do not seem to entirely support this view (Büsselberg et al., 2001, Büsselberg et al., 2003, Johnson et al., 2002, Ramirez et al., 1996). To some extent, synaptic inhibition mediated by GABAA and glycine receptors has already been studied in the adult lamprey (Rovainen, 1983). However, only qualitative data have been provided, and assessments of changes in the timing and intensity components of the breathing-pattern induced by receptor blockades are lacking. Moreover, no information is available on the possible role played by GABAB and GABAC receptors in lamprey respiratory control.

This study was undertaken to quantitatively analyze the respiratory influences exerted by glycine receptors and by different subtypes of GABA receptors in the in vitro brainstem preparation of the adult lamprey. To this end, we investigated the respiratory responses to the bath application of specific antagonists. In addition, specific agonists were also employed to ascertain the effectiveness and selectivity of receptor blockade, as well as to provide further information on the respiratory role of inhibitory neurotransmission. The involvement of endogenously released GABA and glycine was also studied using specific uptake blockers.

Section snippets

Results

The amplitude of the integrated vagal nerve activity was taken as a reliable index of the intensity of respiratory bursts. With regard to the duration of respiratory bursts, these are characteristically very short in the lamprey when compared with those recorded in in vitro preparations from other lower vertebrates or mammals (see, e.g., Bou-Flores and Berger, 2001, Johnson et al., 2002, Lieske et al., 2000, Paton and Richter, 1995, Ramirez et al., 1996). In accordance with previous studies (

Discussion

This study provides a quantitative analysis of the respiratory responses caused by GABA and/or glycine receptor blockade in the adult lamprey. More specifically, the results increase current knowledge with regard to the role of GABAA and glycine receptors in the lamprey respiratory rhythmogenesis. Moreover, they provide data on the respiratory function of GABAB and GABAC receptors. The results support the notion that, at least in vitro, inhibitory synaptic mechanisms are not crucial for

Experimental procedure

All animal care and experimental procedures were conducted in accordance with the Italian legislation and the official regulations of the European Communities Council on the use of laboratory animals (directive 86/609/EEC). The study was approved by the Animal Care and Use Committee of the University of Florence.

Acknowledgments

We are grateful to Dr. Elisabeth Guerin for the English revision of the manuscript. This study was supported by grants from the Ministero dell'Istruzione, Università e Ricerca of Italy.

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