Activation of the putative respiratory rhythm generator in a thick medullary section isolated from bullfrogs

Abstract

The neural basis for breathing in vertebrates arises from central rhythm-generating circuits in the brainstem. Over 30 years of work have focused intensely on the mechanisms of respiratory rhythm generation mainly in neonatal rodents, but how the respiratory rhythm is generated in other vertebrates, including amphibians, remains poorly understood. In frogs, the “lung area” located between cranial nerve (CN) VIII and CN IX, and in the region of the reticularis parvocellularis is thought to generate the rhythm of lung breathing. Although this region is necessary and sufficient for breathing related motor output, the degree to which it functions in isolation, consistent with its role as an endogenous oscillator, is not clear. To address this question, we generated a thick (1.9 ± 0.4 mm) rhythmic medullary slice preparation from the isolated adult frog brainstem. This preparation encompassed the rostral and caudal extent of the vagus nerve root and contained the putative “lung area” and vagal motor neurons. Extracellular output from the vagus nerve was largely silent in these preparations (17 out of 18 experiments). We employed a variety of approaches to increase excitability and elicit fictive breathing as is commonly done in mammals. Elevation of extracellular [K+] (2 out of 3 experiments) or glutamate (3 out of 3 experiments) consistently failed to elicit fictive breathing in the thick slice. However, coadministration of GABA (1 μM Bicuculline) and glycine (3 μM Strychnine) receptor antagonist to block synaptic inhibition reliably initiated and sustained fictive breathing motor patterns (15 out of 17 experiments). Higher dose of these drugs (5 μM Bicuculline, 10 μM Strychnine) led to large and very long bursts that were not consistent with breathing (4 out of 4 experiments). Fictive breaths in the thick slice had similar shape but longer duration than those in the intact brainstem. Activity occurred as single bursts or episodic patterns, similar to the intact brainstem, as well as in vivo. To infer if rhythmic output in the thick slice was generated by the “lung area,” we tested if neuromodulators (NE and 5HT) with well-described effects on the lung motor output in the intact brainstem elicit the same actions in the slice. Indeed, NE decreased (9 out 9 experiments) and 5HT increased (8 out of 8 experiments) frequency of the output, consistent with actions in the intact brainstem. These data suggest the “lung area” can produce respiratory motor output in isolation. However, isolation of the network in vitro somehow enhances tonic inhibition that silences the network. Finally, this preparation provides access to the putative rhythm generating neurons and may lead to new insights into the evolution of the cellular and molecular mechanisms of respiratory rhythm generation. NIH R01NS114514 (JS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.