Oxygen supply and ion homeostasis of the respiratory network in the in vitro perfused brainstem of adult rats.

Abstract

An in vitro arterially perfused medulla preparation of 3- to 8-week-old rats is described in which synchronous rhythmic activity (frequency 4.5 +/- 1.7 cycles/min, burst duration 3.1 +/- 1.1 s, n = 40) was recorded from hypoglossal (XII), vagal (X), or spinal (C1-2) nerves and from different classes of neurons in the region of the ventral respiratory group (VRG). Stimulation of dorsal X nerve rootlets produced a reversible blockade of rhythmic activity. Under steady-state conditions, tissue oxygen (pO2) in the VRG (depth of 600-1600 microns below the ventral surface) fell from 180 to 40 mmHg. Extracellular K+ activity (aKe) in the VRG was about 0.3 mM higher, calcium concentration ([Ca]e) did not differ, and pH (pHe) was about 0.27 units lower than in the perfusion or superfusion solution (with an aKe of 2.2 mM, a [Ca]e of 1.5 mM and a pHe of 7.4). During inspiratory XII nerve discharges, rhythmic increases of aKe by up to 0.8 mM were detected in the VRG. Perfusion of N2-gassed hypoxic solutions (5-10 min) resulted in a tissue anoxia of the VRG and a reversible cessation of rhythmic activity after 2-7 min. Such anoxia was accompanied by a rise of aKe by up to 35 mM, whereas pHe and [Ca]e fell (from mean levels of 7.17 and of 1.5 mM, respectively) by more than 0.2 pH units and 1 mM. Similar observations were made during a 2- to 5-min arrest of the perfusion pump to simulate ischaemia, whereas significantly larger changes in aKe, pHe and [Ca]e were revealed during an "ischaemia" period of 10 min. The results indicate that the rhythmic activity is generated by the functionally intact respiratory network of the VRG in which neurons are under aerobic conditions and ion homeostasis is not impaired. We conclude that the preparation is an appropriate in vitro model for the analysis of the cellular mechanisms for generation of respiratory rhythm and of metabolic perturbations like anoxia and ischaemia in the mature respiratory network.