Persistent sodium current in preBötzinger neurons is not necessary for respiratory rhythm generation in the neonatal mouse?

Abstract

Mammalian breathing is dependent upon the activity of a set of respiratory neurons in the preBotzinger complex (preBotC). Smith and colleagues identified a set of pacemaker neurons whose rhythmicity depends on the activation of a persistent sodium current (INa(P)), in the preBotC (Smith et al. 1991; Del Negro et al. 2002). Since then, many studies have focused on the role of INa(P) in respiratory rhythm generation. Still, it has not been clear whether INa(P) is obligatory for respiratory rhythm generation. INa(P) is widely distributed in neurons in the preBotC and also outside the preBotC (Ptak et al. 2005). Bath application of riluzole, a blocker of INa(P), to brain slices that include the preBotC inhibited the respiratory rhythm; however, this does not necessarily prove a role for INa(P) in the preBotC since riluzole could affect neurons outside preBotC too. The beauty of the new study by Pace et al. is that it directly tested the role of INa(P) in the preBotC in respiratory rhythm generation using carefully controlled drug microinjections (Pace et al. 2007). Muscimol, a GABAA receptor agonist, reversibly inhibits neurons. Microinjecting muscimol into the preBotC reversibly inhibited the respiratory rhythm within 1 min and Pace et al. (2007) used muscimol microinjection to find the appropriate position for microinjecting riluzole. Surprisingly, microinjecting riluzole into the preBotC had no effects on respiratory rhythm generation even when combined with bath application of flufenamic acid (FFA), a calcium-activated non-specific cation current (ICAN) blocker. These results differ from previous experiments using bath application of riluzole and FFA (Pena et al. 2004; Del Negro et al. 2005). Furthermore, Pace et al. (2007) found that the respiratory rhythm was still present after microinjecting another INa(P) blocker, a low concentration solution of TTX, into the preBotC together with bath application of FFA. The frequency of the respiratory rhythm was decreased, but this could be caused by a reduction of action potential amplitude by TTX. Together, these experiments suggest that INa(P) in preBotC neurons is not obligatory for respiratory rhythm generation in neonatal mice. What might explain these differing results on the role of INa(P) in respiratory rhythm generation? One hypothesis is that bath-applied riluzole, or a low concentration solution of TTX, exert their effects on neurons outside the preBotC to inhibit respiratory rhythm generation in the slice preparation. To explore which type of neuron might be the target, Pace et al. (2007) tested serotonergic neurons in the raphe nucleus. They found another surprising result, that microinjecting riluzole into the raphe obscurus with bath application of FFA abolished respiratory rhythm generation reversibly. Thus, it is no surprise that bath application of riluzole and FFA inhibit respiratory rhythm generation. However, this inhibition might not result from the block of INa(P) in preBotC neurons as suggested by previous studies (Pena et al. 2004; Del Negro et al. 2005). In conclusion, this study provides evidence to support the argument that INa(P) in preBotC neurons is not necessary for respiratory rhythm generation in the neonatal mouse. Like other good articles, this one raises many interesting new scientific questions. First, how does riluzole act in the raphe nucleus to abolish the respiratory rhythm? One possibility is that riluzole decreases the excitability of serotonergic neurons, and thus reduces the amplitude of the serotonergic input to the preBotC. Second, is the rhythmic activity of INa(P)-dependent pacemaker neurons in the preBotC inhibited by microinjecting riluzole into the raphe nucleus? If the answer is ‘no’, then the result would demonstrate that the INa(P)-dependent pacemaker activity in the preBotC is not obligatory for respiratory rhythm generation. Certainly, more experiments are needed to clarify these questions.