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Maintenance of eupnea of in situ and in vivo rats following riluzole: A blocker of persistent sodium channels

https://doi.org/10.1016/j.resp.2006.04.018Get rights and content

Abstract

We have proposed a “switching” concept for the neurogenesis of breathing in which rhythm generation by a pontomedullary neuronal circuit for eupnea may be switched to a medullary pacemaker system for gasping. This switch involves activation of conductances through persistent sodium channels. Based upon this proposal, eupnea should continue following a blockade of persistent sodium channels. In situ preparations of the decerebrate, juvenile rat were studied in normocapnia, hypocapnia and hypercapnia. Regardless of the level of CO2 drive, riluzole (1–10 μM), a blocker of persistent sodium channels, caused increases in the frequency and reductions in peak integrated phrenic height. Even 20 μM of riluzole, a concentration four-fold higher than that which eliminates gasping, did not cause a cessation of phrenic discharge. In conscious, rats breathing continued unabated following intravenous administrations of 3–9 mg kg−1 of riluzole. These administrations did cause sedation. We conclude that conductance through persistent sodium channels plays little role in the neurogenesis of eupnea.

Introduction

The mechanisms by which respiratory rhythms are generated within the brainstem remain unclear. Adding complexity to this question of rhythm generation are the multiple rhythms of automatic ventilatory activity, of which eupnea and gasping are two patterns. We have maintained that fundamentally different mechanisms generate eupnea and gasping. Eupnea depends upon synaptic interactions among neurons in a circuit involving both pons and medulla. Embedded in this circuit are medullary neurons with a potential for a pacemaker discharge. Under conditions of brainstem hypoxia, this pacemaker discharge can be released to generate gasping (St.-John, 1990, Rybak et al., 2003a, Rybak et al., 2003b, Paton et al., 2006).

We have validated that a decerebrate, in situ rat preparation can exhibit patterns of rhythmic activity that are very similar to those classically defined as eupnea and gasping of in vivo preparations (St.-John and Paton, 2000, Paton et al., 2006). Using this preparation, we have evaluated the hypothesis that a pacemaker discharge, involving a persistent sodium current, is essential for the neurogenesis of gasping. As a portion of this evaluation, we have found that gasping is totally eliminated, but eupnea little altered, following administration of blockers of persistent sodium channels. Concentrations that eliminated gasping also block persistent sodium currents of in vitro preparations (Paton et al., 2006).

Yet, does the activation of persistent sodium current, and release of medullary mechanisms for gasping, reflect a specific alteration in the environment of the medulla, such as those of hypoxia and/or ischaemia used to induce gasping, or would this activation occur with any generalized increase in respiratory drive? Such a general role during eupnea would require that a blockade of persistent sodium channels should cause marked alterations in phrenic discharge during high respiratory drive such as in hypercapnia and minimal or no alterations at low drive in hypocapnia. The present study was designed to evaluate these possibilities.

Section snippets

Decerebrate, perfused, in situ rats

Thirty-seven perfused preparations of the decerebrate juvenile rat were used. The preparation was identical to that described previously (St.-John and Paton, 2000), with surgical procedures being performed under deep halothane anaesthesia, as assessed by an absence of a withdrawal response to noxious pinching of a paw. Anaesthesia was discontinued following decerebration. Efferent activity of the phrenic nerve was recorded. In 30 of the preparations, recordings were obtained at one level of

Decerebrate, in situ preparations

At normocapnia, variables of phrenic discharge were: TI = 0.69 ± 0.05 s, TE = 4.07 ± 0.25 s and frequency = 13.3 ± 0.8 min−1. For preparations examined at hypocapnia or hypercapnia, variables were similar with the exception that frequency was elevated in hypercapnia (16.7 ± 2.4 min−1, P < 0.05). Phrenic discharge was not systematically examined at several levels of drive in the same preparation, no statistical comparison of peak phrenic height at different levels of CO2 is possible. However, in preparations which

Discussion

The major conclusion of this study is that eupnea continues following administrations of riluzole in concentrations sufficient to cause sedation in unanaesthetized in vivo rats or, as we have reported previously (Paton et al., 2006), to eliminate gasping of in situ preparations. Indeed, the eupneic pattern was significantly distorted only following administrations of riluzole at concentrations four-fold higher than those that totally eliminated gasping. Even at these extreme levels of riluzole,

Acknowledgements

This work was funded by grants from the National Institutes of Health (HL 26091), and the Wellcome Trust. The protocols for use of animals have been approved by the Animal Care and Use Committee of Dartmouth Medical School (USA), the University of Bristol Ethics Committee and the Home Office of the United Kingdom.

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