1. O2-, K+- and pH-sensitive microelectrodes were used to measure extracellular oxygen pressure (P(O2)), K+ activity (aK(o)) and pH (pH(o)) in ventral regions of the medulla oblongata containing respiratory neurons in the in vitro brainstem-spinal cord preparation from 0 to 4-day old rats. 2. The location of respiratory neurons was mapped by extracellular recordings with conventional microelectrodes, or with the reference barrel of ion-sensitive microelectrodes. The major populations of respiratory neurons were distributed in the ventrolateral reticular formation near the nucleus ambiguus at depths of 300-600 mum. In this area, aK(o) baseline increased from 3.2 to 3.8 mM whereas steady-state values of P(O2) and pH(o) fell from 120 to 7 mmHg and from 6.9 to 6.7. respectively. 3. During rhythmic inspiratory discharges recorded with suction electrodes from ventral roots of spinal (C3-C5) and cranial (IX, X, XII) nerves. aK(o) transiently increased by up to 100 muM, and P(O2) fell maximally by 0.4 mmHg. During episodes of non-rhythmic neuronal discharge. aK(o) increased by as much as 0.4 mM and P(O2) decreased by about 10 mmHg. In contrast. no variations in pH(o) could be detected during such activities. 4. Activation of medullary neurons by tetanic electrical stimulation of axonal tracts in the ventrolateral column of the spinal cord at the level of the phrenic motoneuron pool produced aK(o) elevations of up to 5 mM. decreases of P(O2) by up to 50 mmHg, and pH(o) increases by a maximum of 0.07 pH units. These aK(o) and P(O2) transients were reduced by more than 80% during blockade of synaptic transmission with 5 mm manganese (Mn2+) and completely blocked by 1 muM tetrodotoxin (TTX). 5. The tissue P. gradient as well as activity-related decreases of P(O2) were completely abolished after block of oxidative cellular metabolism by addition of 2-10 mm cyanide (CN-) to the bathing solution. 6. Inhibition of the Na+-K+ pump by addition of 3-50 mum ouabain (310 min) caused a reversible increase of aK(o) by 0.8-3 mM, a delayed recovery of stimulus-induced aK(o) elevations, and produced a disturbance of the respiratory rhythm. 7. The sensitivity of the respiratory network to oxygen depletion was tested by superfusing the neuraxis with hypoxic solutions gassed with N2 instead of O2 (5-20 min). The response of the respiratory network to such hypoxic exposure consisted of an initial increase in frequency of respiratory motor output, followed by a depression of respiratory activity that terminated in a reversible loss of the respiratory rhythm. 8. The anoxia-induced disturbance of respiratory rhythm was preceded by a rapid fall of P(O2) to nominally 0 mmHg, an average increase of aK(o) of 1.2 mm, and by a decrease of pH(o) of 0.1-0.4 pH units. During such tissue anoxia, the stimulus-evoked elevations of aK(o) were reduced in amplitude and the recovery of aK(o) to baseline was delayed. 9. Neither baseline levels nor the amplitude or the kinetics of stimulus-induced transients of P(O2) and aK(o) were affected by substitution of the 95% O2, 5% CO2-gassed, HCO3--buffered solution with a 100% O2-gassed, Hepes-buffered saline. In contrast. pH(o) baseline reversibly decreased by 0-2 pH units during a 30 min superfusion with Hepes-buffered saline. Furthermore, stimulus-induced extracellular alkalinizations as well as hypoxia-dependent acidifications were potentiated in Hepes buffer. 10. It is concluded that the oxygen supply of the ventral respiratory network is sufficient to maintain aerobic neuronal metabolism and metabolically driven Na+-K+ pump activity which allows respiratory rhythmogenesis and network function in the in vitro neonatal rat brainstem-spinal cord preparation.
