Nitric oxide as a modulator of central respiratory rhythm in the isolated brainstem of the bullfrog (Rana catesbeiana)

Nitric oxide (NO) is a unique interneuronal neurotransmitter and/or neuromodulator that is involved in a variety of physiological functions within the central nervous system (CNS). In neural tissue, NO is generated from an oxygen-dependent, constitutive NO synthase (NOS) by glutamatergic stimulation of N-methyl-D-aspartate (NMDA) receptors. Recent studies indicate that NO has excitatory effects on breathing within the CNS and mediates a central component of the hypoxic ventilatory reflex in mammals. Because NMDA receptors are important in central respiratory rhythmogenesis, we hypothesized that NO would have significant effects on the central pattern generator (CPG) for breathing in the brainstem. To test this hypothesis, the effects of NO on respiratory-related neural activity were investigated using an in vitro brainstem preparation from North American bullfrogs (Rana catesbeiana). Extracellular recordings of respiratory-related burst activity were made from cranial nerves V, X and XII before and during superfusion of the brainstem with NO-generating compounds, or inhibitors of NO synthesis. Addition of the NO donor, sodium nitroprusside (SNP; 0.1-1.0 mM), or the amino acid precursor for NO synthesis, L-arginine (L-Arg; 0.01-1.0 mM), caused significant increases in respiratory-related burst frequency. Inhibition of NOS with N omega-nitro-L-arginine (L-NA; 5-10 mM), a non-selective NOS inhibitor, caused a significant reduction in burst frequency or reversibly abolished neural activity. Brainstem perfusion with the specific neuronal NOS (nNOS) inhibitor, 7-nitro indazole (7-NI), produced significant, dose-dependent reversible reductions in burst frequency at concentrations of 0.1, 0.5 and 1.0 mM. These results suggest that production of NO, probably via nNOS, provides an excitatory input to the respiratory CPG in the amphibian brainstem. Our results suggest that NO may be a necessary inter- or intracellular messenger for neurotransmission and/or neuromodulation of central respiratory drive to motor effectors in the bullfrog.